CELL

Abstract

The present invention relates to a cell which comprises a chimeric antigen receptor (CAR) and a signal transduction modifying protein, selected from one of the following: (i) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase domain; (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain; (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to fusion proteins and truncated proteins which enable the signalling pathways which are propagated following immune cell activation to be manipulated or modulated.

BACKGROUND TO THE INVENTION

[0002] Adoptive immunotherapy with autologous T cells involves the isolation of T cells from the patient followed by their stimulation, modification and/or expansion ex vivo in order to generate a population of T cells which display anti-tumour specificities. Once re-infused into the patient these cells are capable of recognizing tumour-expressed antigens and mediating tumour rejection.

[0003] This approach has already been shown in a number of trials in different settings to have the potential to be a powerful, effective and long-lasting treatment for cancer. For instance, EBV-driven tumours, such as lymphoproliferative disease following solid organ transplant can be effectively treated by ex vivo expanded EBV specific T-cells.

[0004] A similar therapy for non-viral malignancies involves tumour infiltrating lymphocytes (TILs) which are isolated from resected fragments of tumour and then subjected to stimulation and expansion with autologous tumour samples. Expanded T cell cultures which show tumour reactivity can then be re-infused into the patient.

[0005] Rather than selecting and refining T cell specificities with repeated exposure to antigens, the desired anti-tumour specificity can be conferred onto the T cells through gene modification and the introduction of either a tumour-specific T cell receptor (TCR) or a chimeric antigen receptor (CAR). These cells are expanded ex vivo in order to produce sufficient numbers of cells to achieve meaningful clinical responses within the patient.

[0006] However, the approaches detailed above have limitations. For instance, adoptively transferred T-cells may show limited persistence and expansion in vivo due to insufficient signalling, lack of IL2 or differentiation. By way of further example, adoptively transferred T-cells may succumb to inhibitory stimuli within the tumour microenvironment. For example they may become exhausted, undergo activation induced cell death consequent to over activation, or may cause on-target off-tumour effects.

[0007] Another promising approach to activating therapeutic anti-tumour immunity is the blockade of immune checkpoints. Immune checkpoints refer to the various inhibitory pathways of the immune system that are important for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues.

[0008] It is known that tumours exploit certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumour antigens. Many of the immune checkpoints are initiated by ligand-receptor interactions, meaning that they can be blocked by antibodies or modulated by recombinant forms of ligands or receptors. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies were the first of this class of immunotherapeutics to achieve US Food and Drug Administration (FDA) approval. More recently, blockers of additional immune-checkpoint proteins, such as programmed cell death protein 1 (PD1), have been developed and shown to enhance anti-tumour immunity.

[0009] One problem with the use of immune checkpoint inhibitors is that there are a multitude of inhibitory pathways triggered by a multitude of ligand:receptor interactions. The use of an antibody or a recombinant form of the ligand/receptor will only block one such inhibitory pathway, leaving the possibility open that the tumour can compensate for the specific immune checkpoint block using other molecules.

SUMMARY OF ASPECTS OF THE INVENTION

[0010] The present inventors have developed a system for modulating and/or manipulating signal transduction pathways in immune cells, such as T cells and natural killer (NK) cells.

[0011] Intracellular signalling pathways are initiated and controlled by the reversible post-translational modification of proteins. The present inventors have determined that activating and inhibitory signalling pathways in T cells can be modulated and/or manipulated by fusion proteins or truncated proteins comprising SH2 domains from immediate T-cell signal transduction proteins. In other words, activating and inhibitory signalling pathways in T cells can be modulated and/or manipulated by fusion proteins or truncated proteins comprising SH2 domains from proteins which are capable of binding phosphorylated immunoreceptor tyrosine-based activation motifs (ITAM) or phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM).

[0012] Thus in a first aspect, the present invention provides a cell which comprises a chimeric antigen receptor (CAR) and a signal transduction modifying protein, selected from one of the following:

[0013] (i) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase domain;

[0014] (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain;

[0015] (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain.

[0016] The signal transduction modifying protein may be a truncated protein which comprises a ZAP70 SH2 domain but lacks a ZAP70 kinase domain.

[0017] The signal transduction modifying protein may be a truncated protein which comprises an PTPN6 SH2 but lacks a PTPN6 phosphatase domain.

[0018] The signal transduction modifying protein may be a truncated protein which comprises a SHP-2 SH2 domain but lacks a SHP-2 phosphatase domain.

[0019] The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); and (ii) a phosphatase domain.

[0020] The fusion protein may, for example, comprise a ZAP70 SH2 domain, a PTPN6 or an SHP-2 phosphatase domain.

[0021] The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a kinase domain.

[0022] The fusion protein may comprise an SH2 domain from PTPN6 or SHP-2.

[0023] The fusion protein may comprise a Zap70 kinase domain

[0024] The fusion protein may comprise an AKT or JAK kinase domain.

[0025] The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous signalling domain.

[0026] The fusion protein may comprise an SH2 domain from ZAP70, PTPN6 or SHP-2.

[0027] The heterologous signalling domain may be from a signalling molecule which is not usually activated by an ITAM or ITIM containing receptor.

[0028] The heterologous signalling domain may be a co-stimulatory domain. In this respect, the fusion protein may comprise a CD28, OX40 or 41 BB co-stimulatory domain.

[0029] The heterologous signalling domain may be an inhibitory domain. In this respect, the inhibitory domain may be or comprise the endodomain of CD148 or CD45. Alternatively, the heterologous signalling domain is or comprises the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.

[0030] The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); and (ii) an ITAM-containing domain.

[0031] The fusion protein may comprises a ZAP70 SH2 domain.

[0032] The ITAM-containing domain may be or comprise the endodomain of CD3-Zeta.

[0033] The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) an ITIM-containing domain.

[0034] The fusion protein may comprise an SH2 domain from PTPN6 or SHP-2.

[0035] The ITIM-containing domain may be or comprise the endodomain from PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.

[0036] The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a protease domain.

[0037] The fusion protein may comprise an SH2 domain from ZAP70, PTPN6 or SHP-2.

[0038] The protease domain may be or comprise Tobacco Etch Virus Protease (TeV).

[0039] The cell may also comprises a membrane-tethered transcription factor having a protease cleavage site. Cleavage at the protease cleavage site may release the transcription factor leading to increased expression of a target gene.

[0040] The target gene encodes a cytokine, for example a cytokine selected from the following group: IL-2, IL-7, IL-15 and IL-12.

[0041] In this embodiment, the chimeric antigen receptor (CAR) may be a target CAR which comprises an intracellular protease cleavage site.

[0042] The target CAR may comprise an activatory or co-stimulatory endodomain and cleavage at the protease cleavage site removes the endodomain from the target CAR.

[0043] Alternatively, the target CAR may comprise an inhibitory endodomain and cleavage at the protease cleavage site removes the inhibitory endodomain from the target CAR. The inhibitory endodomain may comprise a CD148 or CD45 endodomain.

[0044] The cell of the present invention may comprise two CARs: an activating CAR comprising an ITAM-containing endodomain; and a target CAR as defined above.

[0045] Alternatively, the cell of the present invention may comprise two CARs: an inhibitory CAR comprising an ITIM-containing endodomain; and a target CAR as defined above.

[0046] In a second aspect, the present invention provides a nucleic acid construct, which comprises:

[0047] a first nucleic acid sequence encoding a chimeric antigen receptor; and

[0048] a second nucleic acid sequence encoding a truncated protein or a fusion protein as defined in connection with the first aspect of the invention.

[0049] The nucleic acid construct may also comprise a third nucleic acid sequence encoding a membrane-tethered transcription factor as defined above.

[0050] The nucleic acid construct may also comprise a third nucleic acid sequence encoding a target CAR as defined above.

[0051] The nucleic acid construct may also comprise a fourth nucleic acid sequence encoding an activating CAR or an inhibitory CAR as defined above.

[0052] In a third aspect, the vector which comprises a nucleic acid construct according to the second aspect of the invention or first and second, and optionally third and/or fouth, nucleic acid sequences as defined above.

[0053] There is also provided a set of vectors which comprises first and second, and optionally third and/or fourth, nucleic acid sequences as defined above.

[0054] The vector or set of vectors may be retroviral or lentiviral vector(s).

[0055] In a fourth aspect, there is provided a pharmaceutical composition comprising a plurality of cells according to the first aspect of the invention.

[0056] In a fifth aspect, there is provided a pharmaceutical composition according to the fourth aspect of the invention for use in treating and/or preventing a disease.

[0057] In a sixth aspect, there is provided method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the fourth aspect of the invention to a subject.

[0058] The method may comprise the following steps:

[0059] (i) isolation of a cell containing sample from a subject;

[0060] (ii) transduction or transfection of the cells with a nucleic acid construct according to the second aspect of the invention, a vector or set of vectors according to the third aspect of the invention; and

[0061] (iii) administering the cells from (ii) to the subject.

[0062] In a seventh aspect there is provided the use of a pharmaceutical composition according to the fourth aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

[0063] The disease may be cancer.

[0064] In an eighth aspect, there is provided a method for making a cell according to the first aspect of the invention, which comprises the step of introducing: a nucleic acid construct according to the second aspect of the invention, a vector or set of vectors according to the third aspect of the invention, into the cell.

[0065] The cell may be from a sample isolated from a subject.

[0066] In a first further aspect, the present invention also provides a fusion protein which comprises: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a heterologous domain.

[0067] The fusion protein may comprise a ZAP70 SH2 domain and an ITAM-containing domain. The ITAM-containing domain may be or comprise the endodomain of CD3-Zeta.

[0068] The fusion protein may comprise a PTPN6 SH2 domain and an ITIM-containing domain. The ITIM-containing domain may be or comprise the endodomain from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.

[0069] The fusion protein may comprise a PTPN6 SH2 domain and fused to a ZAP70 kinase domain.

[0070] The fusion protein may comprise a ZAP70 SH2 domain fused to a PTPN6 kinase domain.

[0071] The fusion protein may comprise: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a heterologous signalling domain.

[0072] The heterologous signalling domain may be from a signalling molecule which is not usually activated by an ITAM containing receptor. The heterologous signalling domain may be or comprise the endodomain of CD28, 41BB or OX40. The heterologous signalling domain may be or comprise the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.

[0073] The fusion protein may comprise: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a kinase domain.

[0074] The kinase domain may be or comprise an AKT or JAK kinase domain.

[0075] The fusion protein may comprise: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a protease domain.

[0076] The protease domain may be or comprise Tobacco Etch Virus Protease (TeV).

[0077] In a second further aspect the present invention provides a truncated protein which comprises the ZAP70 SH2 domain but lacks the ZAP70 kinase domain.

[0078] In a third further aspect the present invention provides a truncated protein which comprises the PTPN6 SH2 domain but lacks the PTPN6 kinase domain.

[0079] The present invention also provides a signalling system comprising:

[0080] (i) a receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signalling domain which comprises a CD3 zeta endodomain; and

[0081] (ii) a fusion protein according to the first further aspect of the invention which comprises a ZAP70 SH2 domain; or a truncated protein according to the second further aspect of the invention;

[0082] wherein binding of antigen to the antigen-binding domain results in binding between the CD3 zeta endodomain and the fusion/truncated protein.

[0083] The present invention also provides a signalling system comprising:

[0084] (i) a receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signalling domain which comprises a PTPN6 binding domain; and

[0085] (ii) a fusion protein according to the first further aspect of the invention which comprises a PTPN6 SH2 domain; or a truncated protein according to the third further aspect of the invention

[0086] wherein binding of antigen to the antigen-binding domain results in binding between the PTPN6 binding domain and the fusion/truncated protein.

[0087] The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0088] In a fourth further aspect the present invention provides a nucleic acid which encodes a fusion protein according to the first further aspect of the present invention or a truncated protein according to the second or third further aspects of the present invention.

[0089] In a fifth further aspect the present invention provides a nucleic acid construct which comprises a nucleic acid sequence encoding a fusion protein which comprises (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain) and a nucleic acid sequence encoding a membrane tethered transcription factor which comprises:

[0090] (i) a membrane tether;

[0091] (ii) a protease recognition site; and

[0092] (iii) a transcription factor.

[0093] In a sixth further aspect the present invention provides a nucleic acid construct which comprises

[0094] (a) a nucleic acid sequence encoding a fusion protein according to the first further aspect of the present invention which comprises a PTPN6 SH2 domain, or a truncated protein according to the third further aspect of the present invention; and

[0095] (b) a nucleic acid sequence encoding a receptor comprising an ITIM containing endodomain.

[0096] In a seventh further aspect the present invention provides a nucleic acid construct which comprises a nucleic acid sequence encoding a fusion protein which comprises (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain) and a nucleic acid sequence encoding a receptor which comprises a protease cleavage site.

[0097] In an eighth further aspect the present invention provides a nucleic acid construct which comprises:

[0098] (a) a nucleic acid sequence encoding a fusion protein which comprises (i) a PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain);

[0099] (b) a nucleic acid sequence encoding a receptor which comprises a protease cleavage site; and

[0100] (c) a nucleic acid sequence encoding a receptor comprising an ITIM containing endodomain.

[0101] The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0102] Suitably, in the nucleic acid construct according to the eighth aspect of the present invention, the nucleic acid sequence (b) may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) which comprises:

[0103] (i) a protease cleavage site between a transmembrane domain and an activating endodomain; or

[0104] (ii) an activating endodomain fused to an inhibitory endodomain via a protease cleavage site.

[0105] In a ninth further aspect the present invention provides a vector comprising a nucleic acid according to the fourth further aspect of the present invention or a nucleic acid construct according to any of fifth to the ninth further aspects of the present invention.

[0106] The vector may be a retroviral vector or a lentiviral vector.

[0107] In a tenth further aspect the present invention provides a cell comprising a fusion protein according to the first further aspect of the present invention or a truncated protein according to the second or third further aspects of the present invention.

[0108] In an eleventh further aspect the present invention provides a cell which comprises (a) a fusion protein according to the first further aspect of the present invention which comprises a PTPN6 SH2 domain, or a truncated protein according to the third further aspect of the present invention; and (b) a receptor comprising an ITIM containing endodomain.

[0109] The cell may be an immune cell, such as a T cell or a natural killer (NK) cell.

[0110] In a twelfth further aspect the present invention provides a cell which comprises a fusion protein which comprises (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain) and a receptor which comprises a protease cleavage site.

[0111] In a thirteenth further aspect the present invention provides a cell which comprises:

(a) a fusion protein which comprises (i) a PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain); (b) a receptor which comprises a protease cleavage site; and (c) a receptor comprising an ITIM containing endodomain.

[0112] The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0113] The receptor (b) may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) which comprises:

[0114] (i) a protease cleavage site between a transmembrane domain and an activating endodomain; or

[0115] (ii) an activating endodomain fused to an inhibitory endodomain via a protease cleavage site.

[0116] In a fourteenth further aspect the present invention provides a cell which comprises a nucleic acid according to the fourth further aspect of the present invention or a nucleic acid construct according to any of the fifth to the ninth further aspects of the present invention.

[0117] In a fifteenth further aspect the present invention provides a pharmaceutical composition comprising a plurality of cells according to any of the tenth to the fourteenth further aspects of the present invention.

[0118] In a sixteenth further aspect the present invention provides a pharmaceutical composition according to the fifteenth further aspect of the present invention for use in treating and/or preventing a disease.

[0119] In a seventeenth further aspect the present invention relates to a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according the fifteenth further aspect to a subject.

[0120] The method may comprise the following steps:

[0121] (i) isolation of a T cell or NK cell containing sample from a subject;

[0122] (ii) transduction or transfection of the T cells or NK cells with a nucleic acid according to any of the fourth to the ninth further aspects of the present invention or a vector according to the tenth further aspect of the present invention; and

[0123] (iii) administering the T cells or NK cells from (ii) to the subject.

[0124] In an eighteenth further aspect the present invention relates to the use of a pharmaceutical composition according to the fifteenth further aspect of the present invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

[0125] The disease may be cancer.

[0126] In a nineteenth further aspect the present invention provides a kit which comprises a nucleic acid according to the fourth further aspect of the present invention or a nucleic acid construct according to any of the fifth to the eighth further aspects of the present invention or a vector according to the ninth further aspect of the present invention.

[0127] In a twentieth further aspect the present invention relates to a kit which comprises a cell according to any of the tenth to the fourteenth further aspects of the present invention.

[0128] In a twenty-first further aspect the present invention relates to a method for making a cell according to any of the tenth to the fourteenth further aspects of the present invention, which comprises the step of introducing: a nucleic acid sequence according to any of the fourth to the eighth further aspects of the present invention or the vector according to the ninth further aspect of the present invention, into the cell.

[0129] The cell may be from a sample isolated from a subject.

[0130] Yet further aspect of the invention are summarised in the following paragraphs:

[0131] A1. A truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) but lacks a kinase domain.

[0132] A2. A truncated protein according to paragraph A1, which comprises the ZAP70 SH2 domain but lacks the ZAP70 kinase domain.

[0133] B1. A truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain.

[0134] B2. A truncated protein according to paragraph 131, which comprises the PTPN6 SH2 domain but lacks the PTPN6 phosphatase domain.

[0135] B3. A truncated protein according to paragraph 131, which comprises the SHP-2 SH2 domain but lacks the SHP-2 phosphatase domain.

[0136] C1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); and (ii) a phosphatase domain.

[0137] C2. A fusion protein according to paragraph C1, which comprises a ZAP70 SH2 domain.

[0138] C3. A fusion protein according to paragraph C1 or C2, which comprises a PTPN6 or SHP-2 phosphatase domain.

[0139] D1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a kinase domain.

[0140] D2. A fusion protein according to paragraph D1, which comprises an SH2 domain from PTPN6 or SHP-2.

[0141] D3. A fusion protein according to paragraph D1 or D2, which comprises a Zap70 kinase domain

[0142] D4. A fusion protein according to paragraph D1 or D2, which comprises an AKT or JAK kinase domain.

[0143] E1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous signalling domain.

[0144] E2. A fusion protein according to paragraph E1, which comprises an SH2 domain from ZAP70, PTPN6 or SHP-2.

[0145] E3. A fusion protein according to paragraph E1 or E2, wherein the heterologous signalling domain is from a signalling molecule which is not usually activated by an ITAM or ITIM containing receptor.

[0146] E4. A fusion protein according to paragraph E1, E2 or E3, wherein the heterologous signalling domain is a co-stimulatory domain.

[0147] E5. A fusion protein according to paragraph E4 which comprises a CD28, OX40 or 41 BB co-stimulatory domain.

[0148] E6. A fusion protein according to paragraph E1, E2 or E3, wherein the co-stimulatory domain is an inhibitory domain.

[0149] E7. A fusion protein according to paragraph E6, wherein the inhibitory domain comprises the endodomain of CD148 or CD45.

[0150] E8. A fusion protein according to paragraph E6, wherein the heterologous signalling domain is or comprises the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.

[0151] F1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); and (ii) an ITAM-containing domain.

[0152] F2. A fusion protein according to paragraph F1, which comprises a ZAP70 SH2 domain.

[0153] F3. A fusion protein according to paragraph F1 or F2, wherein the ITAM-containing domain is or comprises the endodomain of CD3-Zeta.

[0154] G1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) an ITIM-containing domain.

[0155] G2. A fusion protein according to paragraph G1, which comprises an SH2 domain from PTPN6 or SHP-2.

[0156] G3. A fusion protein according to paragraph G1 or G2, wherein the ITIM-containing domain is or comprises the endodomain from PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.

[0157] H1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a protease domain.

[0158] H2. A fusion protein according to paragraph H1, which comprises an SH2 domain from ZAP70, PTPN6 or SHP-2.

[0159] H3. A fusion protein according to paragraph H1 or H2, wherein the protease domain is or comprises Tobacco Etch Virus Protease (TeV).

[0160] I1. A nucleic acid sequence which encodes a truncated protein according to any of paragraphs A or B, or a fusion protein according to any of paragraphs C, D, E, F, G or H.

[0161] J1. A nucleic acid construct which comprises a nucleic acid sequence according to paragraph I and a nucleic acid sequence encoding a chimeric antigen receptor

[0162] J2. A nucleic acid construct which comprises a nucleic acid sequence according to paragraph I and a nucleic acid sequence encoding a membrane-tethered transcription factor having a protease cleavage site.

[0163] J3. A nucleic acid construct which comprises a nucleic acid sequence according to paragraph I and a nucleic acid sequence encoding a target CAR which comprises an intracellular protease cleavage site.

[0164] K1. A vector comprising a nucleic acid sequence according to paragraph I or a nucleic acid construct according to paragraphs J.

[0165] L1. A cell which comprises a truncated protein according to any of paragraphs A or B, or a fusion protein according to any of paragraphs C, D, E, F, G or H.

[0166] M1. A cell which comprises a fusion protein according to any of paragraphs H and a membrane-tethered transcription factor with a protease cleavage site.

[0167] M2. A cell according to paragraph M1, wherein cleavage at the protease cleavage site releases the transcription factor leading to increased expression of a target gene.

[0168] M3. A cell according to paragraph M2, wherein the target gene encodes a cytokine.

[0169] M4. A cell according to paragraph M3, wherein the cytokine is selected from the following group: IL-2, IL-7, IL-15 and IL-12.

[0170] M5. A cell which comprises a fusion protein according to any of paragraphs H and a target receptor (CAR) which comprises an intracellular protease cleavage site.

[0171] M6. A cell according to claim M5, wherein the target CAR comprises an activatory or co-stimulatory endodomain and cleavage at the protease cleavage site removes the endodomain from the target CAR.

[0172] M7. A cell according to claim M5, wherein the target CAR comprises an inhibitory endodomain and cleavage at the protease cleavage site removes the inhibitory endodomain from the target CAR.

[0173] M8. A cell according to paragraph M7, wherein the inhibitory endodomain comprises a CD148 or CD45 endodomain.

[0174] M9. A cell which comprises a fusion protein according to any of paragraphs H and two CARs: an activating CAR comprising an ITAM-containing endodomain; and a target CAR as defined in any of paragraphs M5 to M8.

[0175] M10. A cell which comprises a fusion protein according to any of paragraphs H and two CARs: an inhibitory CAR comprising an ITIM-containing endodomain; and a target CAR as defined in any of paragraphs M5 to M8.

[0176] The aspects of the present invention described above enable T cell signalling pathways to be modulated and altered by, for example, the mechanisms described in Table 1.

TABLE-US-00001 TABLE 1 Application of signal modulation Type Mechanism Application Blocking signal ZAP70, SHP-2 or PTPN6 are Truncated ZAP70, SHP-2 or PTPN6 truncated - keeping SH2 domain competes with wild-type full-length alone ZAP70, SHP-2 or PTPN6. Since this does not signal, it will inhibit activation. Applications include, for example, with ZAP70 when a very strong activation signal is deleterious, or with PTPN6 or SHP-2 when the effect of an inhibitory signal e.g. PD1/PDL1 needs to be reduced. Crosswire signal ZAP70 SH2 fused to PTPN6/SHP- In this embodiment, a ZAP70 SH2 is 2 phosphatase, or PTPN6/SHP-2 fused to the phosphatase from SH2 fused to ZAP70 kinase for PTPN6/SHP-2, or the other way round, instance. i.e. the PTPN6/SHP-2 SH2 domain is fused with the ZAP70 kinase domain. When the T-cell receives an inhibitory signal, it interprets it as an excitatory signal or vice versa. Amplified signal ZAP70 fused to further ITAM A single phospho-ITAM or ITIM leads to domains or PTPN6/SHP-2 fused a concatenation of ITAMs or ITIMs to further ITIM domains. leading to augmented signal or increased sensitivity to antigen. Bypass signal ZAP70 SH2 or PTPN6/SHP-2 SH2 In this embodiment, a "non- fused with e.g. CD28, 41BB physiological" signal can be attached to endodomains or AKT kinase the ITAM/ITM pathway. In this way an domain, a JAK kinase domain ITAM/ITIM signal can lead to a co- etc. stimulatory signal, or a signal such as AKT or a cytokine type signal Transcriptional signal ZAP70 SH2 or PTPN6/SHP-2 SH2 In this embodiment, a transcriptional fused to protease domain along signal is transmitted upon immune with co-expression of a receptor activation or inhibition. Such a membrane tethered signal can, for example, result in the transcription factor with a expression of a particular cytokine upon liberating protease cleavage site T-cell activation or inhibition. Castration signal ZAP70 SH2 domain or In this embodiment, activation or PTPN6/SHP-2 SH2 domain fused inhibition of a receptor results in to a protease domain; a inhibition or activation of another reciprocal receptor has a receptor protease cleavage site

DESCRIPTION OF THE FIGURES

[0177] FIG. 1 (a)--Diagram of immediate T-cell activation pathways. T-cell receptor activation results in phosphorylation of ITAMs. Phosphorylated ITAMs are recognized by the ZAP70 SH2 domains. Upon recognition, ZAP70 is recruited to the juxta-membrane region and its kinase domain subsequently phosphorylates LAT. Phosphorylated LAT is subsequently recognized by the SH2 domains of GRAP, GRB2 and PLC-.gamma.. (b)--Diagram of immediate T-cell inhibition pathways. Activation of an inhibitory immune-receptor such as PD1 results in phosphorylation of ITIM domains. These are recognized by the SH2 domains of PTPN6. Upon recognition, PTPN6 is recruited to the juxta-membrane region and its phosphatase domain subsequently de-phosphorylates ITAM domains inhibiting immune activation.

[0178] FIG. 2--Diagram of a blocking signal system--a) A truncated ZAP70 which does not comprise a kinase domain is over-expressed. Consequently, it competes with full-length ZAP70 for ITAMs and reduces ITAM signalling. (b) A truncated PTPN6 which does not comprise a phosphatase domain is over-expressed, competing for full-length PTPN6 reducing ITIM signalling.

[0179] FIG. 3--Diagram of a crosswire signal system: (a) ZAP70 SH2 is fused to PTPN6 phosphatase, hence acts to dampen ITAM phosphorylation; (b) PTPN6 SH2 is fused to ZAP70 kinase resulting in paradoxical activation in response to an inhibitory signal.

[0180] FIG. 4--Diagram of an amplified signal system: (a) full-length ZAP70 has a CD3 Zeta endodomain attached to its amino terminus so a cascade of ITAMs assembles. (b) full-length PTPN6 has PD1 endodomain attached to its amino terminus so a cascade of ITIMs assembles.

[0181] FIG. 5--Diagram of examples of a bypass signal system: (a) ZAP70 fused with CD28 endodomain; (b) ZAP70 fused with 41BB endodomain; (c) ZAP70 fused with AKT kinase; (d) PTPN6 SH2 domain is fused with 41 BB endodomain

[0182] FIG. 6--Diagram of an illustrative transcriptional signal system: a) A ZAP-TeV fusion is co-expressed with a membrane-tethered transcription factor which can be released from the membrane by cleavage of its TeV recognition motif. This is shown co-expressed with a CD19 CAR. Hence, upon recognition of CD19 on a target cell, the T-cell becomes activated and in addition, the transcription factor becomes active. (b) An alternative system using a PTPN6-TeV fusion instead. Here the CAR consists of an ITIM-bearing endodomain. Hence, upon recognition of CD19 by the CAR, the transcription factor becomes active but this is independent of T-cell activation.

[0183] FIG. 7--Diagram of a castration signal system: two CARs are shown--one which recognizes CD19 and is activating and one which recognizes CD33 and is inhibiting--these specificities are for illustration only (a) AND NOT signal castration; here an SH2-Tev fusion protein is recruited to activated ITIM CAR upon its activation. This results in cleavage of ITAMs from an activating CAR which is constructed such that a TeV cleavage site connects the transmembrane-domain to the ITAM domain. Hence, the activating CAR is inhibited. (b) AND signal castration: Here, an SH2-Tev fusion protein is recruited to an ITIM CAR upon its activation. This results in release of a phosphatase domain from an activating CAR which is constructed so that a phosphatase is connected to its carboxy-terminus via a TeV cleavage domain. This results in release of constitutive inhibition, allowing the CAR to activate in the presence of cognate antigen.

[0184] FIG. 8--Several fusions of different SH2 domains and AKT kinase domain were constructed: ZAP-AKT, GRAP-AKT, GRB-AKT and PLC-.gamma..

[0185] FIG. 9--(a) Phospho-AKT staining of T-cells transduced with the different SH2/AKT fusions with and without activation with the mitogenic antibody OKT3. (b) Phospho-AKT staining of T-cells transduced with ZAP-AKT fusion, an improved ZAP-AKT fusion where ZAP and AKT are connected via a flexible linker, and a control ZAP-AKT where R190K substitution removes ability of ZAP to bind ITAMs. T-cells were either stimulated with OKT3 or not-stimulated with OKT3. The facs plots are overlaid over that of non-transduced T-cells.

[0186] FIG. 10--(a) Phospho-AKT blot of T-cells activated with increasing amounts of OKT3. (b) Microscopy of ZAP-AKT or control T-cells unstimulated, stimulated with just OKT3 or stimulated with both OKT3 and IL-2. ZAP-AKT T-cells stimulated with just OKT3 resemble non-transduced T-cells stimulated with both OKT3 and IL2.

[0187] FIG. 11--(a) Implementation of direct TeV transcriptional switch. A CD19 CAR's endodomain is replaced with the TeV protease. A membrane tethered VP16/GAL4 transcription factor is also co-expressed. A Luciferase reporter detects VP16/GAL5 activity. (b) Implementation with ZAP-TeV. A standard CD19 CAR is co-expressed with ZAP-TeV fusion along with the membrane tethered transcription factor.

[0188] FIG. 12--Activity of ZAP-TeV based transcriptional switches and control expressing T-cells after exposure to CD19 negative (left), or CD19 positive (right) targets. Activity is measured by light output after adding Luciferase. In order the conditions tested are: (a) aCD19 CAR co-expressed with ZAP-TeV; (b) aCD19 CAR co-expressed with inactive (R190K); (c) aCD19 CAR co-expressed with ZAP-TeV and the membrane tethered transcription factor; (d) aCD19 CAR co-expressed with inactive (R190K) ZAP-TEV co-expressed with the membrane tethered transcription factor; (e) aCD19 CAR/TeV fusion co-expressed with the membrane-tethered transcription factor; (f) constitutively active GAL4/VP16 transcription factor.

[0189] FIG. 13--(a) Generalized architecture of a CAR: A binding domain recognizes antigen; the spacer elevates the binding domain from the cell surface; the trans-membrane domain anchors the protein to the membrane and the endodomain transmits signals. (b) to (d): Different generations and permutations of CAR endodomains: (b) initial designs transmitted ITAM signals alone through Fc.epsilon.R1-.gamma. or CD3.zeta. endodomain, while later designs transmitted additional (c) one or (d) two co-stimulatory signals in cis.

[0190] FIG. 14--Illustrative protein sequences of the present invention

[0191] FIG. 15--PD-1 signal blockade using truncated SHP-1 (PTPN6) or truncated SHP-2 PBMC cells were cotransduced with PD1 and either CAR alone (FMC63); or a bicistronic construct containing CAR and truncated SHP-1, or CAR and truncated SHP-2. These cells were co-cultured for 48 hours with SupT1 cells transduced with CD19, PDL1 or both and IFN.gamma. release measured by ELISA.

[0192] FIG. 16--PD-1 signal hijack using a fusion of SHP-2 SH2 domains and Zap70 kinase PBMC cells were cotransduced with PD1 and either CAR alone (FMC63); or a bicistronic construct containing CAR and a fusion protein comprising SHP-2 SH2 domains and the ZAP70 kinase. These cells were co-cultured in a 1:1 ratio for 24 hours with SupT1 cells transduced with CD19 or PDL1. IFN.gamma. release was measured by ELISA (A) and killing of SupT1 cells was quantified by FACS (B).

DETAILED DESCRIPTION

[0193] Protein

[0194] The present invention provides a truncated protein which comprises an SH2 domain.

[0195] The present invention also provides a fusion protein comprising (i) an SH2 domain; and (ii) a heterologous domain.

[0196] The SH2 domain may be from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM).

[0197] An example of a protein which binds an ITAM is ZAP70. Examples of proteins which bind ITIMs include PTPN6 and SHP-2

[0198] The fusion protein of the invention therefore comprises an SH2 domain and at least one further domain which is not present in a wild-type protein from which the SH2 domain was derived.

[0199] SRC Homology 2 (SH2) Domain

[0200] Intracellular signalling pathways are initiated and controlled by the reversible post-translational modification of proteins including phosphorylation, ubiquitinylation and acetylation.

[0201] SH2 domains are modular protein domains that serve as adaptors and mediate protein-protein interactions by binding to phosphorylated peptides in their respective protein binding partners, often cell surface receptors. SH2 domains typically bind a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein, and SH2 domains represent the largest class of known pTyr-recognition domains

[0202] Although SH2 domains lack any intrinsic catalytic activity they are frequently coupled to independent catalytic domains and thus, in response to a specific input signal, serve to localize these catalytic domains so particular sub-cellular locations or to the vicinity of appropriate substrates, activators or inhibitors. In addition SH2 domains can also be found linked to adaptor protein domains and so can serve in the formation of large multi-protein complexes.

[0203] Zeta-Chain-Associated Protein Kinase 70 (ZAP70)

[0204] ZAP70 is a protein normally expressed near the surface membrane of T cells and natural killer cells. It is part of the T cell receptor (TCR), and plays a critical role in T-cell signalling. Its molecular weight is 70 kDa, and is composed of 2 N-terminal SH2 domains and a C-terminal kinase domain. It is a member of the protein-tyrosine kinase family.

[0205] The earliest step in T cell activation is the recognition of a peptide MHC-complex on the target cell by the TCR. This initial event causes the close association of Lck kinase with the cytoplasmic tail of CD3-zeta in the TCR complex. Lck then phosphorylates tyrosine residues in the cytoplasmic tail of CD3-zeta which allows the recruitment of ZAP70. ZAP70 is an SH2 containing kinase that plays a pivotal role in T cell activation following engagement of the TCR. Tandem SH2 domains in ZAP70 bind to the phosphorylated CD3 resulting in ZAP70 being phosphorylated and activated by Lck or by other ZAP70 molecules in trans. Active ZAP70 is then able to phosphorylate downstream membrane proteins, key among them the linker of activated T cells (LAT) protein. LAT is a scaffold protein and its phosphorylation on multiple residues allows it to interact with several other SH2 domain-containing proteins including Grb2, PLC-g and Grap which recognize the phosphorylated peptides in LAT and transmit the T cell activation signal downstream ultimately resulting in a range of T cell responses. This process is summarized in FIG. 1.

[0206] Human ZAP70 protein has the UniProtKB accession number P43403. This sequence is 619 amino acids in length and is shown as SEQ ID NO: 1.

TABLE-US-00002 ZAP70 amino acid sequence (SEQ ID NO: 1) MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARIT SPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFG SVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVR LIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMK YLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGK WPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMA FIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL ASKVEGPPGSTQKAEAACA

[0207] The fusion protein of the invention may comprise a ZAP70 SH2 domain. The truncated protein of the invention may comprise or consist of a ZAP70 SH2 domain. In this respect, the fusion or truncated protein may comprise or consist of the sequence shown as SEQ ID NO: 2.

TABLE-US-00003 ZAP70 complete SH2 domain (SEQ ID NO: 2) MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHP

[0208] ZAP70 has two SH2 domains at the N-terminal end of the sequence, at residues 10-102 and 163-254 of the sequence shown as SEQ ID No. 1. The truncated protein or fusion protein of the invention may therefor comprise one or both of the sequences shown as SEQ ID No. 3 and 4.

TABLE-US-00004 ZAP70 SH2 1 (SEQ ID NO: 3) FFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHF PIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPC ZAP70 SH2 2 (SEQ ID NO: 4) WYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYL ISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEAC

[0209] The fusion protein may comprise a variant of SEQ ID NO: 2, 3 or 4 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence has the required properties. In other words, the variant sequence should be capable of binding to the phosphorylated tyrosine residues in the cytoplasmic tail of CD3-zeta which allow the recruitment of ZAP70.

[0210] Methods of sequence alignment are well known in the art and are accomplished using suitable alignment programs. The % sequence identity refers to the percentage of amino acid or nucleotide residues that are identical in the two sequences when they are optimally aligned. Nucleotide and protein sequence homology or identity may be determined using standard algorithms such as a BLAST program (Basic Local Alignment Search Tool at the National Center for Biotechnology Information) using default parameters, which is publicly available at http://blast.ncbi.nlm.nih.gov. Other algorithms for determining sequence identity or homology include: LALIGN (http://www.ebi.ac.ukfTools/psadalign/and http://www.ebi.ac.uk/Tools/psadalign/nucleotide.html), AMAS (Analysis of Multiply Aligned Sequences, at http://www.compbio.dundee.ac.uk/Software/Amas/amas.html), FASTA (http://www.ebi.ac.ukfTools/sss/fasta/), Clustal Omega (http://www.ebi.ac.ukfTools/msa/clustalo/), SIM (http://web.expasy.org/sim/), and EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss needle/nucleotide.html).

[0211] In certain embodiments, the fusion protein may comprise the ZAP70 SH2 domain and the ZAP70 kinase domain. For example, the fusion protein may comprise the sequence shown as SEQ ID NO: 1 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0212] Tyrosine-Protein Phosphatase Non-Receptor Type 6 (PTPN6)

[0213] PTPN6 is also known as Src homology region 2 domain-containing phosphatase-1 (SHP-1). It is a member of the protein tyrosine phosphatase family.

[0214] The N-terminal region of PTPN6 contains two tandem SH2 domains which mediate the interaction of PTPN6 and its substrates. The C-terminal region contains a tyrosine-protein phosphatase domain.

[0215] PTPN6 is capable of binding to, and propagating signals from, a number of inhibitory immune receptors or ITIM containing receptors. Examples of such receptors include, but are not limited to, PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 and KIR3DL3.

[0216] Human PTPN6 protein has the UniProtKB accession number P29350. This sequence is 595 amino acids in length and is shown as SEQ ID NO: 5.

TABLE-US-00005 PTPN6 amino acid sequence (SEQ ID NO: 5) MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVT HIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVL SDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIE EASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFES LQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMT TREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPL DNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPII VHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQ TEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHA KASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK

[0217] The fusion protein of the invention may comprise a PTPN6 SH2 domain. The truncated protein of the invention may comprise or consist of a PTPN6 SH2 domain. In this respect, the fusion or truncated protein may comprise or consist of the sequence shown as SEQ ID NO: 6.

TABLE-US-00006 PTPN6 SH2 complete domain (SEQ ID NO: 6) MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVT HIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVL SDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIE EASGAFVYLRQPYY

[0218] PTPN6 has two SH2 domains at the N-terminal end of the sequence, at residues 4-100 and 110-213 of the sequence shown as SEQ ID No. 5. The truncated protein or fusion protein of the invention may therefor comprise one or both of the sequences shown as SEQ ID No. 3 and 4.

TABLE-US-00007 PTPN6 SH2 1 (SEQ ID NO: 7) WFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIR IQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL PTPN6 SH2 2 (SEQ ID No. 8) WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPG SPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYL RQPY

[0219] The fusion protein may comprise a variant of SEQ ID NO: 6, 7 or 8 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence has the required properties. In other words, the variant sequence should be capable of binding to the phosphorylated tyrosine residues in the cytoplasmic tail of at least one of PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3 which allow the recruitment of PTPN6.

[0220] In certain embodiments, the fusion protein may comprise the PTPN6 SH2 domain and the PTPN6 phosphatase domain. For example, the fusion protein may comprise the sequence shown as SEQ ID NO: 5 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0221] SHP-2

[0222] SHP-2, also known as PTPN11, PTP-1D and PTP-2C is is a member of the protein tyrosine phosphatase (PTP) family. Like PTPN6, SHP-2 has a domain structure that consists of two tandem SH2 domains in its N-terminus followed by a protein tyrosine phosphatase (PTP) domain. In the inactive state, the N-terminal SH2 domain binds the PTP domain and blocks access of potential substrates to the active site. Thus, SHP-2 is auto-inhibited. Upon binding to target phospho-tyrosyl residues, the N-terminal SH2 domain is released from the PTP domain, catalytically activating the enzyme by relieving the auto-inhibition.

[0223] Human SHP-2 has the UniProtKB accession number P35235-1. This sequence is 597 amino acids in length and is shown as SEQ ID NO: 9.

TABLE-US-00008 SHP-2 amino acid sequence (SEQ ID NO: 9) MTSRRWFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGA VTHIKIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKY PLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLS VRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKN PMVETLGTVLQLKQPLNTTRINAAEIESRVRELSKLAETTDKVKQGFWEE FETLQQQECKLLYSRKEGQRQENKNKNRYKNILPFDHTRVVLHDGDPNEP VSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQENS RVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHDYTLRE LKLSKVGQALLQGNTERTVWQYHFRTWPDHGVPSDPGGVLDFLEEVHHKQ ESIVDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQM VRSQRSGMVQTEAQYRFIYMAVQHYIETLQRRIEEEQKSKRKGHEYTNIK YSLVDQTSGDQSPLPPCTPTPPCAEMREDSARVYENVGLMQQQRSFR

[0224] The fusion protein of the invention may comprise a SHP-2 SH2 domain. The truncated protein of the invention may comprise or consist of a SHP-2 SH2 domain. In this respect, the fusion or truncated protein may comprise or consist of the first SH2 domain of SHP-2, for example comprising amino acids 6-102 of SEQ ID NO. 9 or the second SH2 domain of SHP-2, for example comprising amino acids 112-216 of SHP-2. The fusion or truncated protein may comprise or consist of the sequence shown as SEQ ID NO: 10, 11 or 12.

TABLE-US-00009 SHP-2 first SH2 domain (SEQ ID NO: 10) WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIK IQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPL SHP-2 second SH2 domain (SEQ ID No. 11) WFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESN DGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQ LKQPL SHP-2 both SH2 domains (SEQ ID No. 12) WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIK IQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCA DPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGD DKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVET LGTVLQLKQPL

[0225] The fusion protein may comprise a variant of SEQ ID NO: 10, 11 or 12 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence capable of binding an ITIM-containing domain. For example, the variant sequence may be capable of binding to the phosphorylated tyrosine residues in the cytoplasmic tail of PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.

[0226] Heterologous Domain

[0227] As used herein, the term `heterologous domain` refers to any protein domain which is not present in:

[0228] i) wild type ZAP70 (see SEQ ID NO: 1) for fusion proteins comprising a ZAP70 SH2 domain;

[0229] ii) wild type PTPN6 (see SEQ ID NO: 5) for fusion proteins comprising a PTPN6 SH2 domain; or

[0230] iii) wild-type SHP-2 (see SEQ ID No. 9) for fusion proteins comprising a SHP-2 SH2 domain.

[0231] The heterologous domain may be or be derivable from (e.g. part of) a different protein from ZAP70, SHP-2 or PTPN6.

[0232] Alternatively the fusion protein may comprise a fusion of ZAP70 SH2 domain and a domain from PTPN6, such as the PTPN6 kinase domain. By the same token the fusion protein may comprise a fusion of PTPN6 SH2 domain and a domain from ZAP70, such as the ZAP70 kinase domain.

[0233] Amplified Signal

[0234] The present invention provides a fusion protein which comprises: an SH2 domain from an ITAM-binding protein; and an ITAM-containing domain.

[0235] The present invention also provide a fusion protein which comprises: an SH2 domain from an ITIM-binding protein; and an ITIM-containing domain.

[0236] These "amplified" signalling molecules will amplify an excitatory or inhibitory signal inside an immune cell such as a T cell.

[0237] As shown in FIG. 4, the presence of such molecules will lead to a concatenation of either ITAMs or ITIMs leading to an augmented activatory or inhibitory signal, respectively.

[0238] Amplification of an activatory signal is useful in situations where it is desirable to increase the sensitivity of the immune cell (such as a CAR-T cell) to antigen. This may be the case when, for example, the target antigen is expressed at low levels on the target cells.

[0239] Amplification of an inhibitory systems in situations where it is desirable to reduce or prevent T cell activation. WO2015/075469 describes a panel of "logic gate" chimeric antigen receptor pairs which, when expressed by a cell, such as a T cell, are capable of detecting a particular pattern of expression of at least two target antigens A and B). The "AND NOT gate" described in this application comprises a pair of CARs such that the T cell triggers only when antigen A but not antigen B is present on the target cell. In this AND NOT gate, one CAR (recognising antigen A) has an activating endodomain comprising and ITAM, whereas the other CAR (recognising antigen B) has an inhibitory endodomain which may comprise an ITIM. In the presence of antigen A alone, the presence of unligated inhibitory CAR is insufficient to prevent T cell activation, so activation occurs. However, in the presence of both antigens, areas of membrane form with high concentrations of both activatory and inhibitory CARs. Since both endodomains are concentrated, T-cell activation is prevented or reduced.

[0240] Amplification of the inhibitory signal using an amplified signalling molecule of the present invention could be used in an AND gate to reduce or remove any residual signalling which occurs in the presence of both antigens i.e. from incomplete inhibition of the activatory CAR by the inhibitory CAR.

[0241] ITAM-Containing Domain

[0242] In one embodiment, the fusion protein comprises a ZAP70 SH2 domain and an immunoreceptor tyrosine-based activation motif (ITAM)-containing domain.

[0243] A fusion of full-length ZAP70 with an ITAM containing domain results in a structure which amplifies an activating immune signal. Here, the fusion protein is recruited to a phospho-ITAM immune-receptor endodomain. ZAP70 functions normally to propagate the signal but also provides another set of ITAMs which become phosphorylated and recruit more ZAP70. This may be useful to increase signal strength and may increase sensitivity to low-density antigens, for example. In some embodiments, the fusion may include only the ZAP70 SH2 domain with an ITAM containing endodomain (e.g. the fusion does not contain a ZAP70 kinase domain). In other embodiments, the ratio of ZAP70 catalytic domains (kinase domains) with ITAMs may be varied to affect the kinetics of activation in response to dynamics of the activating receptor interactions with cognate target.

[0244] An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system. The motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/Ix(6-8)YxxL/I).

[0245] ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CD3 and .zeta.-chains of the T cell receptor complex, the CD79-alpha and -beta chains of the B cell receptor complex, and certain Fc receptors. The tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the cell.

[0246] Several proteins are known to contain endodomains with one or more ITAM motifs. Examples of such proteins include the CD3 epsilon chain, the CD3 gamma chain and the CD3 delta chain to name a few. The ITAM motif can be easily recognized as a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I. Typically, but not always, two of these motifs are separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/Ix(6-8)YxxL/1). Hence, one skilled in the art can readily find existing proteins which contain one or more ITAM to transmit an activation signal. Further, given the motif is simple and a complex secondary structure is not required, one skilled in the art can design polypeptides containing artificial ITAMs to transmit an activation signal (see WO 2000063372, which relates to synthetic signalling molecules).

[0247] The ITAM-containing domain may be or comprise a CD3-zeta endodomain. Suitably, the ITAM-containing domain may comprise the sequence shown as SEQ ID NO: 13 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity which retains the capacity to be phosphorylated and recruit ZAP70.

TABLE-US-00010 (CD3-zeta endodomain) SEQ ID NO: 13 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

[0248] By way of example, the fusion protein may be or comprise the sequence shown as SEQ ID NO: 14, which contains a ZAP70-SH2 domain fused to a CD3-zeta endodomain.

TABLE-US-00011 SEQ ID NO: 14 MRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRSGGGGSGGGGSGGGGSGGGGSMPDPAAHLPFFYGSI SRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQL NGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVF DCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYH SSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQ DKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGA AAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVY ESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQID VAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVME MAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAAR NVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRK FSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECP PELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAE AACA

[0249] Suitably, the fusion protein may comprise the sequence shown as SEQ ID NO: 14 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0250] ITIM-Containing Domain

[0251] In one embodiment, the fusion protein comprises a PTPN6 SH2 domain and an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing domain

[0252] A fusion of full-length PTPN6 with an ITIM containing domain results in a structure which amplifies an inhibitory immune signal. Here, the fusion protein is recruited to a phospho-ITIM immune-receptor endodomain. PTPN6 functions normally to propagate the signal but also provides another set of ITIMs which become phosphorylated and recruit more PTPN6. In some embodiments, the fusion may include only the PTPN6 SH2 domain with an ITIM containing endodomain (e.g. the fusion does not contain a PTPN6 phosphatase domain). In other embodiments, the ratio of PTPN6 catalytic domains (phosphatase domains) with ITIMs may be varied to affect the kinetics of activation in response to dynamics of the inhibitory receptor interactions with cognate target.

[0253] An ITIM, is a conserved sequence of amino acids (S/I/V/LxYxxl/V/L) that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing inhibitory receptors interact with their ligand, their ITIM motif becomes phosphorylated by enzymes of the Src kinases, allowing them to recruit PTPN6 via interactions between the PTPN6 SH2 domain and the phosphorylated ITIM domains.

[0254] ITIM containing endodomains include those from CD22, LAIR-1, the Killer inhibitory receptor family (KIR), LILRB1, CTLA4, PD-1, BTLA, for example.

[0255] ITIM endodomains from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 and KIR3DL3 are shown in SEQ ID NO: 15 to 24 respectively

TABLE-US-00012 PDCD1 endodomain SEQ ID NO: 15 CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPC VPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL BTLA4 SEQ ID NO: 16 KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMM EDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGD YENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH LILRB1 SEQ ID NO: 17 LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENL YAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLS GEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPS QEGPSPAVPSIYATLAIH LAIR1 SEQ ID NO: 18 HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRET DTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVAR H CTLA4 SEQ ID NO: 19 FLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPEC EKQFQPYFIPIN KIR2DL1 SEQ ID NO: 20 GNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTV NREDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPN AESRSKVVSCP KIR2DL4 SEQ ID NO: 21 GIARHLHAVIRYSVAIILFTILPFFLLHRWCSKKKENAAVMNQEPAGHRT VNREDSDEQDPQEVTYAQLDHCIFTQRKITGPSQRSKRPSTDTSVCIELP NAEPRALSPAHEHHSQALMGSSRETTALSQTQLASSNVPAAGI KIR2DL5 SEQ ID NO: 22 TGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRT VNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELP NAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI KIR3DL1 SEQ ID NO: 23 KDPRHLHILIGTSVVIILFILLLFFLLHLWCSNKKNAAVMDQEPAGNRTA NSEDSDEQDPEEVTYAQLDHCVFTQRKITRPSQRPKTPPTDTILYTELPN AKPRSKVVSCP KIR3DL3 SEQ ID NO: 24 KDPGNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGN RTVNREDSDEQDPQEVTYAQLNHCVFTQRKITRPSQRPKTPPTDTSV

[0256] The ITIM-containing domain may be or comprise a PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3 endodomain. Suitably, the ITIM-containing domain may comprise the sequence shown any of SEQ ID NO: 15 to 24 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity which retains the capacity to be phosphorylated by Src kinases and amplify an inhibitory immune signal.

[0257] By way of example, the fusion protein may be or comprise the sequence shown as SEQ ID NO: 25, which contains a PTPN6-SH2 domain fused to a PD1 endodomain.

TABLE-US-00013 SEQ ID NO: 25 MTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVF PSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLSGGGGSGGGGSGGGG SGGGGSMVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVR VGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTII HLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGD FVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHF KKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGF WEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDS NIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENS RVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRT LQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLP HAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQ RSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPA MKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKR K

[0258] Suitably, the fusion protein may comprise the sequence shown as SEQ ID NO: 25 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity

[0259] Crosswire Signal

[0260] The present invention provides a fusion protein which comprises: an SH2 domain from an ITAM-binding protein; and a phosphatase domain.

[0261] The present invention also provide a fusion protein which comprises: an SH2 domain from an ITIM-binding protein; and a kinase domain.

[0262] These "crosswire" signalling molecules will reverse an excitatory or inhibitory signal inside an immune cell such as a T cell. When a T-cell receives an excitatory signal, for example following recognition of a target antigen by a CAR, or MHC:peptide by a TCR, the presence of the first type of crosswire molecule will result in the cell interpreting the excitatory signal as an inhibitory signal.

[0263] Dampening down or revising T-cell activation may be useful in a variety of situations, for example, it may be used for CAR-expressing T cells where there is a high level of expression of the target antigen on the target cell. It may be used to prevent T-cell over-activation which may lead to T cell exhaustion and/or activation-induced cell death. Preventing a T-cell becoming activated too much or too quickly may also prevent or reduce pathological side effects of CAR-T cell treatment such as cytokine release syndrome (CRS).

[0264] The reverse situation is when a T-cell receives an inhibitory signal, for example following ligation of PD1, and the presence of the second type of crosswire molecule results in the cell interpreting the inhibitory signal as an excitatory signal.

[0265] Reducing or reversing T-cell inhibition will help the cell overcome the inhibitory stimuli within the hostile tumour microenvironment and should therefore increase T-cell persistence and expansion in vivo.

[0266] In one embodiment, the fusion protein comprises a PTPN6 SH2 domain and a ZAP70 kinase domain. In another embodiment the present fusion protein comprises a ZAP70 SH2 domain fused to a PTPN6 kinase domain.

[0267] In embodiments relating to a ZAP70 SH2 domain fused to the phosphatase domain from PTPN6, when the T cell receives an excitatory signal it interprets it as an inhibitory signal because the PTPN6 phosphatase domain is recruited to the activated ITAM via the ZAP70 SH2 domain.

[0268] In embodiments relating to a PTPN6 SH2 domain fused to the kinase domain from ZAP70, when the T cell receives an inhibitory signal it interprets it as an excitatory signal because the ZAP70 kinase domain is recruited to the activated ITIM via the PTPN6 domain. A fusion between PTPN6 SH2 domain and ZAP70 kinase domain will result in competition for phosphorylated ITIMs by wild-type PTPN6 blocking inhibitory signals, but in addition will transmit a paradoxical activation signal. This may have application in over-coming checkpoint blockade signals in a tumour microenvironment.

[0269] The sequence of human ZAP70 kinase, PTPN6 phosphatase and SHP-2 phosphatase domains domains are shown as SEQ ID NO: 26, 27 and 28 respectively.

TABLE-US-00014 ZAP70 kinase domain SEQ ID NO: 26 DPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKV LKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGG PLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLV NRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRS DVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYA LMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA PTPN6 phosphatase domain SEQ ID NO: 27 FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRD SNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQEN SRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLR TLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESL PHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRA QRSGMVQTEAQYKFIYVAIAQFIETTKKKL SHP-2 phosphatase domain SEQ ID NO: 28 WEEFETLQQQECKLLYSRKEGQRQENKNKNRYKNILPFDHTRVVLHDGDP NEPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQ ENSRVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHDYT LRELKLSKVGQALLQGNTERTVWQYHFRTWPDHGVPSDPGGVLDFLEEVH HKQESIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKT IQMVRSQRSGMVQTEAQYRFIYMA

[0270] The ZAP70 kinase domain, PTPN6 phosphatase domain or SHP-2 phosphatase domain may be or comprise the sequence shown as SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, respectively; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity which retains the capacity to phosphorylate or dephosphorylate downstream proteins in the same manner as the wild-type kinase/phosphatase domains.

[0271] Examples of fusion protein comprising a PTPN6 SH2 domain fused to a ZAP70 kinase domain; a ZAP70 SH2 domain fused to a PTPN6 kinase domain; and a SHP-2 SH2 domain fused to a ZAP70 kinase domain are shown as SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID No. 61, respectively.

TABLE-US-00015 PTPN6 SH2 domain fusion: ZAP70 kinase domain SEQ ID NO: 29 MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVT HIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVL SDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIE EASGAFVYLRQPYYSGGGGSDPEELKDKKLFLKRDNLLIADIELGCGNFG SVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVR LIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMK YLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGK WPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMA FIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL ASKVEGPPGSTQKAEAACA ZAP70 SH2 domain fusion: PTPN6 phosphatase domain SEQ ID NO: 30 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHPSGGGGSGGGGSGGGGSGGGG SFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGR DSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQE NSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKL RTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQES LPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVR AQRSGMVQTEAQYKFIYVAIAQFIETTKKKL dual SH2 domains from SHP-2 fused to ZAP70 kinase domain SEQ ID NO. 61 WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIK IQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCA DPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGD DKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVET LGTVLQLKQPLNTTRINPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTL NSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNL LIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREA QIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSN VAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALG ADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQ KPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFL TVEQRMRACYYSL

[0272] The fusion protein may be or comprise the sequence shown as SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID No. 61 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0273] Heterologous Signalling Domain

[0274] The present fusion protein may comprise (i) a ZAP70, PTPN6 or SHP-2 SH2 domain; and (ii) a heterologous signalling domain.

[0275] As used herein, the term "heterologous signalling domain" refers to a signalling domain which is not present in the wild type ZAP70, PTPN6 or SHP-2 protein. As such, where the fusion protein comprises a ZAP70 SH2 domain, it comprises a signalling domain which is not the ZAP70 kinase domain. Alternatively, where the fusion protein comprises a PTPN6 SH2 domain, it comprises a signalling domain which is not the PTPN6 phosphatase domain.

[0276] Bypass Signal

[0277] The heterologous signalling domain may be from a signalling molecule which is not usually activated by an ITAM containing receptor. In other words, the heterologous signalling domain may be from a signalling molecule which is not involved in the propagation of immunological signal 1 following the binding of antigen to the TCR. Immunological signal 1 is sufficient to trigger T-cell killing of cognate target cells but does not fully activate the T-cell to proliferate and survive.

[0278] In one embodiment of this aspect of the invention, the present invention provides a fusion protein which comprises (i) an SH2 domain from a protein which binds an ITAM; and (ii) a heterologous signalling domain.

[0279] A fusion between, for example, ZAP70 and another signaling molecule not typically activated with an ITAM containing receptor may act to bypass signal from one pathway into another. One example is co-stimulation. A fusion between ZAP70 and the endodomain of CD28 may transmit a CD28 co-stimulatory signal as well as an ITAM activatory signal. Similarly, a fusion between ZAP70 and the endodomain of 41 BB or OX40 may transmit a 41 BB or OX40 co-stimulatory signal. Other pathways may also be recruited, for instance a fusion between ZAP70 and AKT kinase domain may result in transmission of an AKT signal upon ITAM phosphorylation. Other examples might include Kinase domain from JAK. In this way, a T-cell may interpret a simple antigen recognition signal as transmitting a co-stimulatory or even a cytokine type signal.

[0280] Such fusion proteins may be useful, for example, in approaches where repeated ex vivo stimulations of T cells can result in populations which lack costimulatory surface antigens and which have limited proliferative capacity in vivo resulting in limited persistence and efficacy. The loss of costimulatory surface antigens leading to activation of T cells solely through the TCR has been linked to a greater degree of activation induced cell death which would negatively impact in vivo efficacy and persistence. The effect can be reversed by the activation of surface-expressed 4-1 BB and OX40 demonstrating that costimulation can prevent activation induced cell death and can support greater expansion of tumour specific T cells.

[0281] In another embodiment of this aspect of the invention, the present invention provides a fusion protein which comprises (i) an SH2 domain from a protein which binds an ITIM; and (ii) a heterologous signalling domain.

[0282] For example, a PTPN6 SH2 domain or SHP-2 SH2 domain may be fused to a co-stimulatory endodomain so a T-cell interprets an inhibitory signal as a co-stimulatory one.

[0283] The heterologous signalling domain may be from, for example, CD28, 41 BB or OX40.

[0284] CD28 provides a potent co-stimulatory signal--namely immunological signal 2, which triggers T-cell proliferation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.

[0285] 41BB (CD137) is a type 2 transmembrane glycoprotein belonging to the TNF superfamily, expressed on activated T cells. Crosslinking of 41BB enhances T cell proliferation, IL-2 secretion survival and cytolytic activity.

[0286] OX40 (CD134) is a secondary co-stimulatory molecule, expressed after 24 to 72 hours following activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is following their activation. Expression of OX40 is dependent on full activation of the T cell; without CD28, expression of OX40 is delayed and of fourfold lower levels. Signalling through OX40 is required for prolonged T cell survival following initial activation and proliferation.

[0287] The CD28, 41BB and OX40 signalling domains (endodomains) are shown as SEQ ID NO: 31, 32 and 33, respectively.

TABLE-US-00016 CD28 endodomain SEQ ID NO: 31 MRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 41BB endodomain SEQ ID NO: 32 MKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL OX40 endodomain SEQ ID NO: 33 MRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0288] The heterologous signalling domain may be or comprise the sequence shown as SEQ ID NO: 31; SEQ ID NO: 32 or SEQ ID NO: 33, respectively, or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0289] The heterologous signalling domain may be or comprise an inhibitory signalling domain.

[0290] For example, the inhibitory signalling domain may comprise the endodomain of CD148 or CD45. CD148 and CD45 have been shown to act naturally on the phosphorylated tyrosines up-stream of TCR signalling.

[0291] CD148 is a receptor-like protein tyrosine phosphatase which negatively regulates TCR signaling by interfering with the phosphorylation and function of PLCy1 and LAT.

[0292] CD45 present on all hematopoetic cells, is a protein tyrosine phosphatase which is capable of regulating signal transduction and functional responses, again by phosphorylating PLC .gamma.1.

[0293] An inhibitory signalling domain may comprise all of part of a receptor-like tyrosine phosphatase. The phospatase may interfere with the phosphorylation and/or function of elements involved in T-cell signalling, such as PLCy1 and/or LAT.

[0294] The inhibitory signalling domain may be or comprise the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.

[0295] The inhibitory signalling domain may comprise the sequence shown as SEQ ID NO: 34 to 39 or a variant thereof having at least 80% sequence identity.

TABLE-US-00017 ICOS endodomain SEQ ID NO: 34 CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL CD27 endodomain SEQ ID NO: 35 QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP BTLA endodomain SEQ ID NO: 36 RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDN DPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEA PTEYASICVRS CD30 endodomain SEQ ID NO: 37 HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPV AEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVST EHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHT PHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK GITR endodomain SEQ ID NO: 38 QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEK GRLGDLWV HVEM endodomain SEQ ID NO: 39 CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETI PSFTGRSPNH

[0296] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 34 to 39 provided that the sequence provides an effective intracellular signalling domain.

[0297] Suitably, the fusion protein may be or comprise any of the sequences shown as SEQ ID NOs: 40 to 45.

TABLE-US-00018 CD28 endodomain fused to amino-terminus of full-length ZAP (SEQ ID NO: 40) MRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSGGGGSGG GGSGGGGSGGGGSMPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLR QCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFY SRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEA LEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKF LLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVE YLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTL NSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNL LIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREA QIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSN VAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALG ADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQ KPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFL TVEQRMRACYYSLASKVEGPPGSTQKAEAACA 41BB endodomain fused to amino-terminus of full-length ZAP (SEQ ID NO: 41) MKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSGGGGSG GGGSGGGGSGGGGSMPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLL RQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEF YSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGE ALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGK FLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLV EYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDT LNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDN LLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMRE AQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVS NVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKAL GADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYG QKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDF LTVEQRMRACYYSLASKVEGPPGSTQKAEAACA OX40 endodomain fused to amino-terminus of full-length ZAP (SEQ ID NO: 42) MRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKISGGGGSGGGGSGG GGSGGGGSMPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRS LGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPD GLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAI ISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPR KEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLK ADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGY TPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADI ELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQ LDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELL HQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSY YTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKK MKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQR MRACYYSLASKVEGPPGSTQKAEAACA CD28 endodomain fused to the amino-terminus of PTPN6 SH2 domain. (SEQ ID NO: 43) MRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSGGGGSGG GGSGGGGSGGGGSMVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQG DFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQ DRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRE SLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSL TDLVEHFKKTGIEEASGAFVYLRQPY 41BB endodomain fused to the amino-terminus of PTPN6 SH2 domain (SEQ ID NO: 44) MKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSGGGGSG GGGSGGGGSGGGGSMVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQ GDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVL QDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVR ESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDS LTDLVEHFKKTGIEEASGAFVYLRQPY OX40 endodomain fused to the amino-terminus of PTPN6 SH2 domain (SEQ ID NO: 45) MRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKISGGGGSGGGGSGG GGSGGGGSMVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLS VRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGT IIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQP GDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVE HFKKTGIEEASGAFVYLRQPY

[0298] Suitably, the fusion protein may comprise the sequence shown as any of SEQ ID NOs: 40 to 45 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity

[0299] Kinase Domain

[0300] The heterologous signalling domain may be a kinase domain. For example, the heterologous signalling domain may comprise an AKT kinase domain or a JAK kinase domain.

[0301] Akt, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration.

[0302] Following activation of the TCR, T cells secrete IL2 which supports survival and proliferation. However this secretion is transient and T cells that are activated and expanded in vitro become dependent on exogenous IL2 for survival. By increasing AKT phosphorylation following ITAM phosphorylation associated with TCR or CAR activation, the dependence of activated T cells on exogenous IL2 may be reduced or removed and their proliferation and survival enhanced.

[0303] The Akt kinase domain is shown as SEQ ID NO: 46.

TABLE-US-00019 Akt kinase domain SEQ ID NO: 46 AEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKI LKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYA NGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENL MLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAV DWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLL SGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQVT SETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA

[0304] The heterologous signalling domain may be or comprise the sequence shown as SEQ ID NO: 46, or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity provided that the sequence provides an effective kinase domain.

[0305] By way of example, the fusion protein may be or comprise the any of the sequences shown as SEQ ID NO: 47 to 49 and 62 which contain a ZAP70-SH2 domain fused directly to an Akt kinase domain, a ZAP70-SH2 domain fused to an Akt kinase domain via a linker a ZAP70 mutated to be non-functional and fused to an Akt kinase domain; and a dual SHP-2 SH2 domain fused to an Akt kinase domain, respectively.

TABLE-US-00020 ZAP70-SH2 domain fused directly to an Akt kinase domain SEQ ID NO: 47 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHPAEEMEVSLAKPKHRVTMNEF EYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENR VLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRA RFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGI KDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFY NQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAK EIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITIT PPDQDDSMECVDSERRPHFPQFSYSASGTA ZAP70-SH2 domain fused to an Akt kinase domain via a linker SEQ ID NO: 48 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHPSGGGGSGGGGSGGGGSGGGG SAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMK ILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEY ANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLEN LMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRA VDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSL LSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQV TSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGT A ZAP70 mutated to be non-functional and fused to an Akt kinase domain SEQ ID NO: 49 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLKPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHPAEEMEVSLAKPKHRVTMNEF EYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENR VLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRA RFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGI KDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFY NQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAK EIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITIT PPDQDDSMECVDSERRPHFPQFSYSASGTA dual SHP-2 SH2 domain fused to an Akt kinase domain SEQ ID No. 62 WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIK IQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCA DPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGD DKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVET LGTVLQLKQPLNTTRINAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTAL KYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDY LHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTP EYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILM EEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVW QHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDS ERRPHFPQFSYSASGTA

[0306] The fusion protein may comprise the sequence shown as any of SEQ ID NO: 47 to 49 or 62 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0307] Janus kinase (JAK) is a family of intracellular, nonreceptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. The four JAK family members are: Janus kinase 1 (JAK1); Janus kinase 2 (JAK2); Janus kinase 3 (JAK3); and Tyrosine kinase 2 (TYK2).

TABLE-US-00021 Kinase containing domain of JAK2 SEQ ID NO: 50 RNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLDKAHRNY SESFFEAASMMSKLSHKHLVLNYGVCVCGDENILVQEFVKFGSLDTYLKK NKNCINILWKLEVAKQLAWAMHFLEENTLIHGNVCAKNILLIREEDRKTG NPPFIKLSDPGISITVLPKDILQERIPWVPPECIENPKNLNLATDKWSFG TTLWEICSGGDKPLSALDSQRKLQFYEDRHQLPAPKWAELANLINNCMDY EPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRIGALGFSGAFEDRD PTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEE HLRDFEREIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDY LQKHKERIDHIKLLQYTSQICKGMEYLGTKRYIHRDLATRNILVENENRV KIGDFGLTKVLPQDKEYYKVKEPGESPIFWYAPESLTESKFSVASDVWSF GVVLYELFTYIEKSKSPPAEFMRMIGNDKQGQMIVFHLIELLKNNGRLPR PDGCPDEIYMIMTECWNNNVNQRPSFRDLALRVDQIRDNM

[0308] Protease Domain

[0309] The present invention also provides a fusion protein which comprises (i) an SH2 domain from a protein which binds an ITAM or ITIM-containing protein and (ii) a protease domain.

[0310] The protease domain may be any protease which is capable of cleaving at a specific recognition sequence. As such the protease domain may be any protease which enables the separation of a single target polypeptide into two distinct polypeptides via cleavage at a specific target sequence.

[0311] The protease domain may be a Tobacco Etch Virus (TeV) protease domain.

[0312] TeV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TeV cleavage site is ENLYFQ\S (where `\` denotes the cleaved peptide bond). Mammalian cells, such as human cells, do not express endogenous TeV protease.

[0313] Accordingly, the TeV cleavage recognition site is shown as SEQ ID NO: 51.

[0314] SEQ ID NO: 51--Tev cleavage site

[0315] ENLYFQS

[0316] The TeV protease domain is shown as SEQ ID NO: 52.

TABLE-US-00022 SEQ ID NO: 52 SLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNN GTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFRE PQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCG SPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGW RLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQ

[0317] Accordingly, the protease domain may be or comprise the sequence shown as SEQ ID NO: 52, or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity provided that the sequence provides an effective protease function.

[0318] By way of example, the fusion protein may be or comprise the sequence shown as SEQ ID NO: 53 or 54, which contains a ZAP70-SH2 domain fused to a TEV protease sequence or a PTPN6-SH2 domain fused to a TEV protease sequence; respectively.

TABLE-US-00023 SEQ ID NO: 53 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL SLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL KEACPNSSASNASGAAAPTLPAHPSTLTHPSGGGGSGGGGSGGGGSGGGG SSLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRN NGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFR EPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQC GSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWV SGWRLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQ SEQ ID NO: 54 MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVT HIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVL SDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIE EASGAFVYLRQPYYSGGGGSSLFKGPRDYNPISSTICHLTNESDGHTTSL YGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDM IIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTF PSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSV PKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMSKPEEPFQPVKEAT QLMNELVYSQ

[0319] The fusion protein may comprise the sequence shown as SEQ ID NO: 53 or 54; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0320] The SH2 domain and heterologous domain of the fusion protein may be separated by a linker in order to spatially separate the SH2 domain and the heterologous domain.

[0321] Transcriptional Signal

[0322] A fusion protein which comprises a protease, as described in the previous section, may be co-expressed in a cell with a membrane-tethered protein having a protease cleavage site. Cleavage of the membrane-tethered protein at the protease site will release the membrane-distal part of the protein.

[0323] The membrane tethered protein may, for example, be a membrane-tethered transcription factor. When cleavage occurs, the transcription is released from its tether and free to transit to the nucleus.

[0324] A fusion between ZAP70 SH2 or PTPN6 SH2 domain and a protease domain will result in membrane-proximal recruitment of the protease following ITAM or ITIM phosphorylation, respectively.

[0325] Phosphorylation of ITAM or ITIM domains results in recruitment of the ZAP70 SH2 or PTPN6 SH2 fused with the protease domain, respectively, to the membrane-proximal area. This results in the transcription factor being cleaved from its tether and transferred to the nucleus. This may have many applications: for example upon activation the T-cell may be programmed to express transcription factors which act to prevent the T-cell from differentiating. For instance, upon activation the T-cell may be programmed to express a cytokine such as IL2, IL7 or IL15 which may act to stimulate proliferation and survival of the T-cell, or IL12 which may convert a hostile tumour microenvironment to one which more favours immune rejection of a tumour.

[0326] In particular, there is provided a cell which co-expresses:

[0327] (i) a fusion protein comprising an SH2 domain from a protein which binds a phosphorylated ITAM; and

[0328] (ii) a membrane tethered transcription factor

[0329] wherein the transcription factor, when released from the membrane tether, increases the expression of IL2, I17 and/or ID 5 in the cell.

[0330] There is also provided a cell which co-expresses:

[0331] (i) a fusion protein comprising an SH2 domain from a protein which binds a phosphorylated ITIM; and

[0332] (ii) a membrane tethered transcription factor

[0333] wherein the transcription factor, when released from the membrane tether, increases the expression of ID 2 in the cell.

[0334] Protease Recognition Site

[0335] The protease recognition site may be any amino acid sequence which enables the protease domain of the fusion protein to specifically cleave the membrane tethered transcription factor between the membrane tether and the transcription factor. For example, in one embodiment the protease domain is a TeV protease domain and the protease recognition site is a TeV protease recognition site.

[0336] Membrane Tether

[0337] The membrane tether may be any sequence, signal or domain which is capable of localising the transcription factor and protease recognition site proximal to a membrane. For example, the membrane tether may be a myrsitylation signal or a transmembrane domain.

[0338] Suitably, a transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (U.S. Pat. No. 7,052,906 B1 describes synthetic transmembrane components).

[0339] The transmembrane domain may be derived from CD28, which gives good stability.

[0340] Transcription Factor

[0341] The transcription factor may be any transcription factor chosen to stimulate a desired response following phosphorylation of the relevant ITAM or ITIM motifs.

[0342] The transcripton factor can be natural or artificial. Artificial transcription factors may be derived from, for example, TALENs, zinc-finger assemblies or CrispR/CAS9, the latter co-expressed with a guide mRNA.

[0343] Preferably, the transcription factor will contain a nuclear localization signal to aid its transportation to the nuclease following cleavage by the protease domain.

[0344] By way of example, nucleic acid sequence (ii) (which encodes a protein comprising a membrane tethered transcription factor which comprises: (i) a membrane tether; (ii) a protease recognition site; and (iii) a transcription factor) may encode a protein which consists of or comprises the sequence shown as SEQ ID NO: 55, which contains a RQR8 domain; a CD4-Endotox1 transmembrane domain, a TEV protease recognition site and a VP16-GAL4 transcription factor.

TABLE-US-00024 SEQ ID NO: 55 MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTN VSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQA ERMAQIKRVVSEKKTAQAPHRFQKTCSPISGGGGSENLYFQMPKKKRKVA PPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSA PYGALDMADFEFEQMFTDALGIDEYGGSGGGSMQILVASDATMKLLSSIE QACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVE SRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVT DRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTV

[0345] Suitably, the protein may comprise the sequence shown as SEQ ID NO: 55; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity

[0346] Receptor

[0347] The present invention further provides a nucleic acid construct which comprises (a) a nucleic acid sequence encoding a fusion protein according to the first aspect of the present invention which comprises a PTPN6 SH2 domain, or a truncated protein according to the third aspect of the present invention; and (b) a nucleic acid sequence encoding a receptor comprising an ITIM containing endodomain.

[0348] Castration Signal

[0349] A fusion protein which comprises a protease, as described above, may be co-expressed in a cell with a target receptor which comprises an intracellular protease cleavage site. Cleavage of the target receptor at the protease site will release an intracellular, membrane-distal part of the target receptor.

[0350] The target receptor may, for example, be a T-cell receptor (TCR), or a chimeric antigen receptor (CAR).

[0351] The receptor may comprise an activatory or co-stimulatory endodomain positioned at the end of the intracellular part of the protein. Cleavage at the protease cleavage site then removes the activatory or co-stimulatory endodomain from the target CAR, reducing or preventing target receptor-mediated T cell activation.

[0352] Alternatively, the target receptor may comprise an inhibitory endodomain positioned at the end of the intracellular part of the protein. Cleavage at the protease cleavage site then removes the inhibitory endodomain from the target CAR, "switching-on" the potential for target receptor mediated T cell activation.

[0353] The inhibitory endodomain may, for example, comprise a CD148 or CD45 endodomain or an ITIM-containing endodomain from a protein such a PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.

[0354] By way of example, the target receptor may comprise the sequence shown as SEQ ID NO: 56, which contains a CAR against CD33 containing an ITIM endodomain from PD-1.

TABLE-US-00025 SEQ ID NO: 56 MAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIY FNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQP EDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSR SEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVS SISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQ DAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPE DCRPRGSVKGTGLDFACDIYVGVVGGLLGSLVLLVWVLAVICSRAARGTI GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYAT IVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0355] Suitably, the protein may comprise the sequence shown as SEQ ID NO: 56; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0356] Where the receptor comprises a protease cleavage site between a transmembrane domain and an activating endodomain, the castration signal fusion protein may be used to inhibit the receptor. For instance, a first CAR might be constructed whereby its endodomain is separated from the transmembrane domain by a protease cleavage site. A second CAR recognizing a different antigen might comprise of an ITIM containing endodomain. Recognition of the cognate antigen of the second receptor would result in recruitment of the castration signal fusion protein to the membrane and subsequent cleavage at the protease recognition site. Such cleavage would separate the activating endodomain from the first receptor and prevent activation and signal propagation from said receptor.

[0357] This would result in an "AND NOT" type logic gate where a sustained signal would be transmitted only if the first CAR was activated in isolation (i.e. when the first CAR bound its cognate antigen but the second CAR did not bind its cognate antigen). Such `logic gates` may be useful, for example, because it is relatively rare for the presence (or absence) of a single antigen to effectively describe a cancer, which can lead to a lack of specificity. Targeting antigen expression on normal cells leads to on-target, off-tumour toxicity. In some cancers, a tumour is best defined by presence of one antigen (typically a tissue-specific antigen) and the absence of another antigen which is present on normal cells. For example, acute myeloid leukaemia (AML) cells express CD33. Normal stem cells express CD33 but also express CD34, while AML cells are typically CD34 negative. Targeting CD33 alone to treat AML is associated with significant toxicity as it depletes normal stem cells. However, specifically targeting cells which are CD33 positive but not CD34 positive would avoid this considerable off-target toxicity.

[0358] Potential pairs of antigens for such an `AND NOT` gate are shown in Table 2.

TABLE-US-00026 TABLE 2 Normal cell which Antigen expressed by normal Disease TAA expresses TAA cell but not cancer cell AML CD33 stem cells CD34 Myeloma BCMA Dendritic cells CD1c B-CLL CD160 Natural Killer cells CD56 Prostate PSMA Neural Tissue NCAM cancer Bowel A33 Normal bowel HLA class I cancer epithelium

[0359] By way of example, the receptor which comprises a protease cleavage site between a transmembrane domain and an activating endodomain may be the sequence shown as SEQ ID NO: 57, which contains a CAR against CD19 with a cleavable CD3-zeta endodomain.

TABLE-US-00027 SEQ ID NO: 57 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDI SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGS EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRCRHR RRQAERMAQIKRVVSEKKTAQAPHRFQKTCSPISGGGGSENLYFQMRRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR

[0360] Suitably, the receptor may comprise the sequence shown as SEQ ID NO: 57; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0361] Where the receptor comprises an activating endodomain fused to an inhibitory endodomain via a protease cleavage site, a castration signal fusion protein can be used to activate artificial signalling domains. For instance, a first CAR might be constructed whereby its endodomain comprises an activating endodomain fused to an inhibitory endodomain via a protease cleavage site. A second CAR recognizing a different antigen might comprise of an ITIM containing endodomain. Recognition of the cognate antigen of the second receptor would result in recruitment of the castration signal fusion protein to the membrane and subsequent cleavage of inhibitory endodomain from the activating endodomain of the first receptor. Cleavage, and thus separation, of the inhibitory domain from the activating domain would allow activation of the first CAR following antigen binding and hence activation of signalling via the first receptor.

[0362] This would result in an "AND" type CAR logic gate where productive signalling would occur only if both the first and second receptors were activated. Such `logic gates` are useful, for example, because most cancers cannot be differentiated from normal tissues on the basis of a single antigen. Hence, considerable "on-target off-tumour" toxicity occurs whereby normal tissues are damaged by the therapy. For some cancers, targeting the presence of two cancer antigens may be more selective and therefore effective than targeting one. For example, B-chronic lymphocytic leukaemia (B-CLL) is a common leukaemia which is currently treated by targeting CD19. This treats the lymphoma but also depletes the entire B-cell compartment such that the treatment has a considerable toxic effect. B-CLL has an unusual phenotype in that CD5 and CD19 are co-expressed. By targeting only cells which express CD5 and CD19, it would be possible to considerably reduce on-target off-tumour toxicity.

[0363] Potential pairs of antigens for such an `AND` logic gate are shown in Table 3.

TABLE-US-00028 TABLE 3 Cancer Type Antigens Chronic Lymphocytic Leukaemia CD5, CD19 Neuroblastoma ALK, GD2 Glioma EGFR, Vimentin Multiple myeloma BCMA, CD138 Renal Cell Carcinoma Carbonic anhydrase IX, G250 T-ALL CD2, N-Cadherin Prostate Cancer PSMA, hepsin (or others)

[0364] By way of example, the receptor which comprises an activating endodomain fused to an inhibitory endodomain via a protease cleavage site may be the sequence shown as SEQ ID NO: 58, which contains a CAR against CD19 with a CD3-zeta endodomain and a cleavable CD148 endodomain.

TABLE-US-00029 SEQ ID NO: 58 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDI SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGS EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPRENLYFQMAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSF SQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAA ELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFI ATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQD YGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPD TTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIE NENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLI YQNTTAMTIYENLAPVTTFGKTNGYIA

[0365] Suitably, the receptor may comprise the sequence shown as SEQ ID NO: 58; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.

[0366] Blocking Signal

[0367] The present invention provides a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) but lacks a kinase domain

[0368] For example, the truncated protein may comprise the ZAP70 SH2 domain but lack the ZAP70 kinase domain. In other words, the present invention provides a truncated protein which: (i) comprises the sequence shown as SEQ ID NO: 2 but does not comprise the sequence shown as SEQ ID NO: 26.

[0369] Over-expression of the ZAP70 SH2 domain results in competition with full-length/wild-type ZAP70. Since the truncated ZAP70 cannot propagate signals, signal transmission is reduced in proportion to the ratio between wild-type ZAP70 and the truncated protein. This may be useful to reduce strength of T-cell activation for instance to prevent T-cell over-activation which can result in T-cell exhaustion, activation induced cell death and in a clinical setting can result in cytokine storms.

[0370] The present invention also provide a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain

[0371] For example, the truncated protein may comprise the PTPN6 SH2 domain but lack the PTPN6 phosphatase domain. In other words, the present invention provides a truncated protein which: (i) comprises the sequence shown as SEQ ID NO: 6 but does not comprise the sequence shown as SEQ ID NO: 27.

[0372] In this case, ITIM signalling can be reduced in proportion to the ratio between wild-type PTPN6 and the truncated protein. This may be useful to reduce inhibitory signals such as PD1 signalling. This may have application when T-cells are targeting a tumour which over-expresses PDL1 (or similar inhibitory receptors) to evade immune rejection.

[0373] The use of a blocking signal or a cross-wire signal as described above, offers a significant advantage over traditional immune checkpoint blockade approaches which typically block a single ligand/receptor interaction, such as PD-L1/PD1, with an antibody. As explained above, the inhibitory immune receptor class contains many members with redundancies and expression patterns which fluctuate with T-cell state. The use of an antibody or a recombinant ligand/receptor may effectively block one inhibitory receptor, but will not affect inhibitory signals transmitted from the rest. Genomic editing of individual inhibitory receptors (Menger et al, Cancer Res. 2016 Apr. 15; 76(8):2087-93) has a similar limitation. Strategies of fusions between individual inhibitory receptors and co-stimulatory domains also suffer from similar limitations (Liu et al, Cancer Res. 2016 Mar. 15; 76(6):1578-90).

[0374] The method of the present invention will block (and depending on the strategy re-interpret) inhibitory signals transmitted via an ITIM. Hence an entire class of inhibitory signals are modulated. A list of inhibitory receptors which signal through ITIMs is provided in Table II of Odorizzi and Wherry (2012) J. Immunol. 188:2957-2965. They include: PD1, BTLA, 2B4, CTLA-4, GP49B, Lair-1, Pir-B, PECAM-1, CD22, Siglec 7, Siglec 9, KLRG1, ILT2, CD94-NKG2A and CD5.

[0375] Nucleic Acid

[0376] In one aspect the present invention provides a nucleic acid which encodes a fusion protein or a truncated protein according to the present invention.

[0377] As used herein, the terms "polynucleotide", "nucleotide", and "nucleic acid" are intended to be synonymous with each other.

[0378] It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

[0379] Nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

[0380] The terms "variant", "homologue" or "derivative" in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

[0381] Nucleic Acid Construct

[0382] In one aspect the present invention provides a nucleic acid construct which co-expresses a truncated protein or fusion protein of the present invention with another protein. The nucleic acid construct may comprise: a nucleic acid sequence encoding a truncated protein or a fusion protein of the present invention; and a nucleic acid encoding another protein.

[0383] The present invention provides a nucleic acid construct which co-expresses a truncated protein or fusion protein of the present invention with a chimeric antigen receptor. The nucleic acid construct may comprise: (i) a nucleic acid sequence encoding a truncated protein or a fusion protein of the present invention; and (ii) a nucleic acid encoding a chimeric antigen receptor.

[0384] The chimeric antigen receptor (CAR) may be an activatory CAR comprising an ITAM-containing endodomain, such as CD3 zeta. The CAR may be an inhibitory CAR comprising a "ligation-off" endodomain, as described in WO2015/075469 which may comprise all or part of the endodomain from a receptor-like tyrosine phosphatase, such as CD148 or CD45. The CAR may be an inhibitory CAR comprising a "ligation-on" endodomain, as described in WO2015/075470 which may comprise an ITIM domain.

[0385] The fusion proteins and truncated proteins of the invention may be used together with a cell expressing a "logic gate" combination of two or more CARs. An OR gate comprises two activatory CARs as described in WO2015/075468. An AND gate comprises an activatory CAR and a "ligation off" inhibitory CAR, as described in WO2015/075469. An AND not comprises an activatory CAR and a "ligation on" inhibitory CAR, as described in WO2015/075470.

[0386] Thus the present invention provides a nucleic acid construct which comprises:

[0387] (i) a nucleic acid sequence encoding a truncated protein or fusion protein of the invention;

[0388] (ii) a first chimeric antigen receptor (CAR); and

[0389] (iii) a second chimeric antigen receptor.

[0390] With reference to the transcription signal aspect of the invention, there is provided a nucleic acid construct which comprises (i) a nucleic acid sequence encoding a fusion protein comprising a SH2 domain; and a protease domain; and (ii) a nucleic acid sequence encoding a membrane tethered transcription factor which comprises: a membrane tether; a protease recognition site; and a transcription factor.

[0391] With reference to the castration signal aspect of the invention, there is provided a nucleic acid construct which comprises (i) a nucleic acid sequence encoding a fusion protein which comprises an SH2 domain and a protease domain (e.g. a TeV domain); and (ii) a nucleic acid sequence encoding a receptor which comprises a protease cleavage site.

[0392] For example, the present invention provides a nucleic acid construct which comprises: (a) a nucleic acid sequence encoding a fusion protein which comprises (i) PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain); (b) a nucleic acid sequence encoding a receptor which comprises a protease cleavage site; and (c) a nucleic acid sequence encoding a receptor comprising an ITIM containing endodomain.

[0393] The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0394] Suitably, the protein encoded by nucleic acid sequence (b) may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) which comprises: (i) a protease cleavage site between a transmembrane domain and an activating endodomain; or (ii) an activating endodomain fused to an inhibitory endodomain via a protease cleavage site.

[0395] Where the nucleic acid construct of the invention, produces discrete polypeptides, such when it coexpresses a fusion protein of the invention and a CAR, it may also comprise a nucleic acid sequence enabling expression of both proteins. For example, it may comprise a sequence encoding a cleavage site between the two nucleic acid sequences. The cleavage site may be self-cleaving, such that when the nascent polypeptide is produced, it is immediately cleaved into the two proteins without the need for any external cleavage activity.

[0396] Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2a self-cleaving peptide, which has the sequence shown:

TABLE-US-00030 SEQ ID NO: 59 RAEGRGSLLTCGDVEENPGP or SEQ ID NO: 60 QCTNYALLKLAGDVESNPGP

[0397] The co-expressing sequence may be an internal ribosome entry sequence (IRES). The co-expressing sequence may be an internal promoter.

[0398] Chimeric Antigen Receptor (CAR)

[0399] CARs, which are shown schematically in FIG. 13, are chimeric type I trans-membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of IgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the IgG1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0400] Early CAR designs had endodomains derived from the intracellular parts of either the .gamma. chain of the Fc.epsilon.R1 or CD3.zeta.. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3.zeta. results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal--namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related OX40 and 41BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

[0401] CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. Lentiviral vectors may be employed. In this way, a large number of cancer-specific T cells can be generated for adoptive cell transfer. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

[0402] CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signalling domain.

[0403] Antigen Binding Domain

[0404] The antigen binding domain is the portion of the CAR which recognizes antigen. Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.

[0405] The antigen binding domain may comprise a domain which is not based on the antigen binding site of an antibody. For example the antigen binding domain may comprise a domain based on a protein/peptide which is a soluble ligand for a tumour cell surface receptor (e.g. a soluble peptide such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand or a receptor for which the binding pair counterpart is expressed on the tumour cell.

[0406] The antigen binding domain may be based on a natural ligand of the antigen.

[0407] The antigen binding domain may comprise an affinity peptide from a combinatorial library or a de novo designed affinity protein/peptide.

[0408] Spacer Domain

[0409] CARs comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

[0410] In aspects of the present invention which require two CARs, the first and second CARs may comprise different spacer molecules. For example, the spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. A human IgG1 spacer may be altered to remove Fc binding motifs.

[0411] All the spacer domains mentioned above form homodimers. However the mechanism is not limited to using homodimeric receptors and should work with monomeric receptors as long as the spacer is sufficiently rigid. An example of such a spacer is CD2 or truncated CD22.

[0412] Since CARs are typically homodimers (see FIG. 13a), cross-pairing may result in a heterodimeric chimeric antigen receptor. This is undesirable for various reasons, for example: (1) the epitope may not be at the same "level" on the target cell so that a cross-paired CAR may only be able to bind to one antigen; (2) the VH and VL from the two different scFv could swap over and either fail to recognize target or worse recognize an unexpected and unpredicted antigen. For the "AND" and "AND NOT" gates described above, the spacer of the first CAR may be sufficiently different from the spacer of the second CAR in order to avoid cross-pairing but sufficiently similar to co-localise. Pairs of orthologous spacer sequences may be employed. Examples are murine and human CD8 stalks, or alternatively spacer domains which are monomeric--for instance the ectodomain of CD2.

[0413] Examples of spacer pairs which co-localise are shown in the following Table:

TABLE-US-00031 Stimulatory CAR spacer Inhibitory CAR spacer Human-CD8aSTK Mouse CD8aSTK Human-CD28STK Mouse CD8aSTK Human-IgG-Hinge Human-CD3z ectodomain Human-CD8aSTK Mouse CD28STK Human-CD28STK Mouse CD28STK Human-IgG-Hinge-CH2CH3 Human-IgM-Hinge-CH2CH3CD4

[0414] Transmembrane Domain

[0415] The transmembrane domain is the sequence of the CAR that spans the membrane.

[0416] A transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (U.S. Pat. No. 7,052,906 B1 describes synthetic transmembrane components).

[0417] The transmembrane domain may be derived from CD28, which gives good receptor stability.

[0418] Activating Endodomain

[0419] The endodomain is the signal-transmission portion of the CAR. It may be part of or associate with the intracellular domain of the CAR. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling may be needed. For example, chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative/survival signal, or all three can be used together.

[0420] Where a CAR comprises an activating endodomain, it may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with that of either CD28 or OX40 or the CD28 endodomain and OX40 and CD3-Zeta endodomain.

[0421] Any endodomain which contains an ITAM motif can act as an activation endodomain in this invention. Suitable endodomains which contain an ITAM motif are described herein.

[0422] Inhibitory Domain

[0423] In embodiments referred to above as the "AND" gate, the first CAR may comprise an activating endodomain fused to an inhibitory endodomain via a protease cleavage site. As such the inhibitory endodomain inhibits T-cell activation by the first CAR in the absence of activation of the second CAR. Upon activation of the second CAR, the ITIM in the endodomain of the second CAR is phosphorylated and the PTPN6/protease domain fusion protein is recruited to the membrane. This results in cleavage of the first CAR between the activating endodomain and inhibitory endodomain, thus enabling T-cell activation.

[0424] The inhibitory endodomains may comprise any sequence which inhibits T-cell signalling by the activating CAR when it is in the same endodomain.

[0425] The inhibitory endodomain may be or comprise a tyrosine phosphatase, such as a receptor-like tyrosine phosphatase. An inhibitory endodomain may be or comprise any tyrosine phosphatase that is capable of inhibiting the TCR signalling when co-localised with the activating endodomain of the CAR. An inhibitory endodomain may be or comprise any tyrosine phosphatase with a sufficiently fast catalytic rate for phosphorylated ITAMs that is capable of inhibiting the TCR signalling when co-localised with the activating endodomain of the CAR.

[0426] Vector

[0427] The present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) or construct(s) according to the present invention. Such a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses the proteins encoded by the nucleic acid sequence or construct.

[0428] The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

[0429] The vector may be capable of transfecting or transducing a T cell.

[0430] Cell

[0431] The present invention also relates to an immune cell comprising the fusion protein, truncated protein, nucleic acid and/or nucleic acid construct of the present invention.

[0432] The cell may be a cytolytic immune cell.

[0433] Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below.

[0434] Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

[0435] Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0436] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0437] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

[0438] Two major classes of CD4+ Treg cells have been described--naturally occurring Treg cells and adaptive Treg cells.

[0439] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

[0440] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

[0441] Natural Killer Cells (or NK cells) are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner.

[0442] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.

[0443] The cells of the invention may be any of the cell types mentioned above.

[0444] T or NK cells expressing the molecules of the invention may either be created ex vivo either from a patient's own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).

[0445] Alternatively, T or NK cells expressing the molecules of the invention may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.

[0446] In all these embodiments, cells are generated by introducing DNA or RNA coding for the receptor component and signalling component by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0447] The cell of the invention may be an ex vivo T or NK cell from a subject. The T or NK cell may be from a peripheral blood mononuclear cell (PBMC) sample. T or NK cells may be activated and/or expanded prior to being transduced with nucleic acid of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

[0448] The T or NK cell of the invention may be made by:

[0449] (i) isolation of a T or NK cell-containing sample from a subject or other sources listed above; and

[0450] (ii) transduction or transfection of the T or NK cells with one or more a nucleic acid sequence(s) according to the invention.

[0451] The T or NK cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.

[0452] The present invention also provides a cell which comprises a fusion protein or a truncated protein of the invention and a chimeric antigen receptor (CAR).

[0453] The chimeric antigen receptor (CAR) may be an activatory CAR comprising an ITAM-containing endodomain, such as CD3 zeta. The CAR may be an inhibitory CAR comprising a "ligation-off" endodomain, as described in WO2015/075469 which may comprise all or part of the endodomain from a receptor-like tyrosine phosphatase, such as CD148 or CD45. The CAR may be an inhibitory CAR comprising a "ligation-on" endodomain, as described in WO2015/075470 which may comprise an ITIM domain.

[0454] The fusion proteins and truncated proteins of the invention may be used together with a cell expressing a "logic gate" combination of two or more CARs. An OR gate comprises two activatory CARs as described in WO2015/075468. An AND gate comprises an activatory CAR and a "ligation off" inhibitory CAR, as described in WO2015/075469. An AND not comprises an activatory CAR and a "ligation on" inhibitory CAR, as described in WO2015/075470.

[0455] Thus the present invention provides a cell which comprises:

[0456] (i) a nucleic acid sequence encoding a truncated protein or fusion protein of the invention;

[0457] (ii) a first chimeric antigen receptor (CAR); and

[0458] (iii) a second chimeric antigen receptor.

[0459] With reference to the transcription signal aspect of the invention, there is provided a cell which comprises (i) a fusion protein comprising an SH2 domain and a protease; and (ii) a membrane tethered transcription factor which comprises: a membrane tether, a protease recognition site; and a transcription factor.

[0460] With reference to the castration signal aspect of the invention there is provided a cell which comprises (i) a fusion protein comprising an SH2 domain and a protease; and (ii) receptor which comprises a protease cleavage site.

[0461] The receptor may, for example be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) which comprises: (i) a protease cleavage site between a transmembrane domain and an activating endodomain; or (ii) an activating endodomain fused to an inhibitory endodomain via a protease cleavage site.

[0462] Composition

[0463] The present invention also relates to a pharmaceutical composition containing a plurality of cells of the invention. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

[0464] Method of Treatment

[0465] The cells of the present invention may be capable of killing target cells, such as cancer cells.

[0466] The cells of the present invention may be used for the treatment of an infection, such as a viral infection.

[0467] The cells of the invention may also be used for the control of pathogenic immune responses, for example in autoimmune diseases, allergies and graft-vs-host rejection.

[0468] The cells of the invention may be used for the treatment of a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

[0469] The cells of the invention may be used to treat: cancers of the oral cavity and pharynx which includes cancer of the tongue, mouth and pharynx; cancers of the digestive system which includes oesophageal, gastric and colorectal cancers; cancers of the liver and biliary tree which includes hepatocellular carcinomas and cholangiocarcinomas; cancers of the respiratory system which includes bronchogenic cancers and cancers of the larynx; cancers of bone and joints which includes osteosarcoma; cancers of the skin which includes melanoma; breast cancer; cancers of the genital tract which include uterine, ovarian and cervical cancer in women, prostate and testicular cancer in men; cancers of the renal tract which include renal cell carcinoma and transitional cell carcinomas of the utterers or bladder; brain cancers including gliomas, glioblastoma multiforme and medullobastomas; cancers of the endocrine system including thyroid cancer, adrenal carcinoma and cancers associated with multiple endocrine neoplasm syndromes; lymphomas including Hodgkin's lymphoma and non-Hodgkin lymphoma; Multiple Myeloma and plasmacytomas; leukaemias both acute and chronic, myeloid or lymphoid; and cancers of other and unspecified sites including neuroblastoma.

[0470] Treatment with the cells of the invention may help prevent the escape or release of tumour cells which often occurs with standard approaches.

[0471] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1--Subjugation of the T Cell Activation Pathway to Augmented or Non-Physiological Signals

[0472] A number of SH2 domains involved in early T cell signal activation were tested to determine whether T cell activation signals could be subjugated or "hijacked", such that when a T cell was activated the signal could be modulated or re-transmitted.

[0473] The inventors generated several chimeric AKT constructs by linking the kinase domain of AKT to SH2 domains from Zap70, Grap, Grb2 and PLCy (FIG. 8).

[0474] In non-transduced (NT) T cells, very low levels of phosphorylation of the endogenous AKT were detectable following treatment with OKT3 to induce cross-linking and activation of the TCR (FIG. 10b:top panel). However, in cells expression the Zap-AKT construct, significant levels of phospho-AKT were observed (FIG. 10b:bottom panel).

[0475] Linker for activation of T cell (LAT) is a downstream target of ZAP70 and is bound by several SH2-containing proteins, such as Grb2, Grap and PLCy. It was anticipated that the SH2 domains from each of these LAT-binders would also allow the activation signal from CD3-zeta to be hijacked. However, this was not the case.

[0476] No TCR-dependent phosphorylation of the AKT kinase domain was observed above levels observed for NT T cells when the AKT kinase domain was linked to the SH2 domains from Grb2, Grap or PLCy (FIG. 9a).

[0477] This demonstrates that this system of T cell signalling hijacking specifically requires the tandem SH2 domain from a very early T cell signalling molecule, such as Zap70 or Tyrosine-protein phosphatase non-receptor type 6 (PTPN6).

Example 2--Transcriptional Control

[0478] The TeV protease was fused to the Zap70 SH2 domain. A membrane-bound transcription factor was also generated as follows: RQR8 was cloned in frame with the VP16/GAL4 transcription factor separated by a TeV cleavage site. This fusion protein allows release of the VP16/GAL4 transcription factor (which contains a nuclear localizing signal) upon TeV cleavage.

[0479] These proteins were both expressed in a T-cell which also expressed a CD19-specific chimeric antigen receptor. To demonstrate that the ZAP70-TeV approach is needed, the transcription factor was co-expressed with a CD19 CAR whose endodomain was replaced by TeV (FIG. 11).

[0480] T-cells were exposed to CD19 negative and positive targets. Transcriptional activation was measured by a Luciferase cassette responsive to GALv/VP16. Only the condition where a standard CD19 CAR was co-expressed with ZAP-TeV and the membrane tethered transcription factor resulted in selective transcriptional activation upon CD19 recognition. The CD19 CAR fused directly to TeV resulted in constitutive transcriptional activation (FIG. 12).

Example 3--PD-1 Signal Blockade Using Truncated SHP-1 (PTPN6) or Truncated SHP-2

[0481] PBMC cells were transduced as shown in the following table:

TABLE-US-00032 Name on FIG. 15 key Description Construct(s) NT Untransduced -- FMC63 Transduced with CD19 CAR only SFG.aCD19_fmc63-HCH2CH3w- CD28tmZw PD1 Transduced with PD1 only pDual-PD1-GFP FMC63 + PD1 Co-transduced with CD19CAR SFG.aCD19_fmc63-HCH2CH3w- and PD1 CD28tmZw and pDual-PD1-GFP FMC63 - Co-transduced with a) bicistronic SFG.aCD19_fmc63-HCH2CH3w- SHP1 + PD1 construct encoding CD19CAR CD28tm-Zeta_w-2A-dualSH2_SHP-1 and truncated SHP1, and b) PD1 and pDual-PD1-GFP FMC63 - Co-transduced with a) bicistronic SFG.aCD19_fmc63-HCH2CH3w- SHP2 + PD1 construct encoding CD19CAR CD28tm-Zeta_w-2A-dualSH2_SHP-2 and truncated SHP1, and b) PD1 and pDual-PD1-GFP

[0482] The cells were co-cultured for 48 hours with SupT1 cells transduced with CD19, PDL1 or both and IFN.gamma. release measured by ELISA. The results are shown in FIG. 15.

[0483] The presence of PDL1 on SupT1 target cells caused a reduction in IFN.gamma. release. There was increased IFN.gamma. release with PBMC which expressed CAR together with the truncated SHP-1 or truncated SHP-2 construct compared with those which expressed CAR alone. This indicates that the truncated SHp-1 and SHP-2 constructs successfully inhibited the PDL1 inhibitory signal from the target cells.

Example 4--PD-1 Signal Hijack Using a Fusion of SHP-2 SH2 Domains and Zap70 Kinase

[0484] PBMC cells were transduced as shown in the following table:

TABLE-US-00033 Name on FIG. 16 key Description Construct(s) NT Untransduced -- FMC63 Transduced with CD19 CAR only SFG.aCD19_fmc63-HCH2CH3w- CD28tmZw PD1 Transduced with PD1 only pDual-PD1-GFP FMC63 + PD1 Co-transduced with CD19CAR SFG.aCD19_fmc63-HCH2CH3w- and PD1 CD28tmZw and pDual-PD1-GFP FMC63 - Co-transduced with a) bicistronic SFG.aCD19_fmc63-HCH2CH3w- SHP2Zap70 + PD1 construct encoding CD19CAR CD28tm-Zeta_w-2A-dualSH2_SHP- and fusion of SHP2 SH2domains 2-Zap70_Kinase and pDual-PD1- and Zap70 kinase, and b) PD1 GFP

[0485] The cells were co-cultured in a 1:1 ratio for 24 hours with SupT1 cells transduced with CD19 or PDL1. IFN.gamma. release was measured by ELISA (FIG. 16A). An increase in IFN-.gamma. production in was seen co-cultures of CAR-SHP2.Zap70+PD1 transduced T cells with PDL1 SupT1 target cells compared with CAR+PD1 transduced T cells.

[0486] A cytotoxicity assay was also conducted in which killing of SupT1 cells was quantified by FACS (FIG. 16B). Near complete killing of PDL1 SupT1 targets was observed in co-cultures of PDL1 positive target cells with CAR-SHP2.Zap70+PD1 transduced T cells. By contrast, killing was not seen with CAR+PD1 alone construct. This indicated that replacing the phosphatase domain of SHP2 with the kinase domain of Zap70 successfully converted the inhibitory PD1 signal to an activatory signal. The SHP-2-Zap70 kinase fusion protein therefore successfully hijacked the inhibitory PDL1-PD1 signal and turned it into a T-cell activation signal.

[0487] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cellular immunology or related fields are intended to be within the scope of the following claims.

Sequence CWU 1

1

631619PRTHomo sapiens 1Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn 275 280 285 Ser Asp Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys 290 295 300 Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser 305 310 315 320 Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn 325 330 335 Leu Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val 340 345 350 Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile 355 360 365 Lys Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met 370 375 380 Arg Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg 385 390 395 400 Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met 405 410 415 Ala Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu 420 425 430 Ile Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly 435 440 445 Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala 450 455 460 Arg Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe 465 470 475 480 Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg 485 490 495 Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn 500 505 510 Phe Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr 515 520 525 Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys 530 535 540 Gly Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys 545 550 555 560 Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp 565 570 575 Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg 580 585 590 Met Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro 595 600 605 Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 610 615 2280PRTArtificial SequenceZAP70 complete SH2 domain 2Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro 275 280 393PRTArtificial SequenceZAP70 SH2 1 3Phe Phe Tyr Gly Ser Ile Ser Arg Ala Glu Ala Glu Glu His Leu Lys 1 5 10 15 Leu Ala Gly Met Ala Asp Gly Leu Phe Leu Leu Arg Gln Cys Leu Arg 20 25 30 Ser Leu Gly Gly Tyr Val Leu Ser Leu Val His Asp Val Arg Phe His 35 40 45 His Phe Pro Ile Glu Arg Gln Leu Asn Gly Thr Tyr Ala Ile Ala Gly 50 55 60 Gly Lys Ala His Cys Gly Pro Ala Glu Leu Cys Glu Phe Tyr Ser Arg 65 70 75 80 Asp Pro Asp Gly Leu Pro Cys Asn Leu Arg Lys Pro Cys 85 90 492PRTArtificial SequenceZAP70 SH2 2 4Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys Leu Tyr 1 5 10 15 Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg Lys Glu 20 25 30 Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val Tyr His 35 40 45 Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro Glu Gly 50 55 60 Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys Leu Lys 65 70 75 80 Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys 85 90 5595PRTHomo sapiens 5Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45 Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His 100 105 110 Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly 115 120 125 Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135 140 Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 145 150 155 160 Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175 Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180 185 190 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr 195 200 205 Leu Arg Gln Pro Tyr Tyr Ala Thr Arg Val Asn Ala Ala Asp Ile Glu 210 215 220 Asn Arg Val Leu Glu Leu Asn Lys Lys Gln Glu Ser Glu Asp Thr Ala 225 230 235 240 Lys Ala Gly Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu Val 245 250 255 Lys Asn Leu His Gln Arg Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly 260 265 270 Lys Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Ser Arg Val Ile 275 280 285 Leu Gln Gly Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala 290 295 300 Asn Tyr Ile Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr 305 310 315 320 Tyr Ile Ala Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp 325 330 335 Gln Met Ala Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg 340 345 350 Glu Val Glu Lys Gly Arg Asn Lys Cys Val Pro Tyr Trp Pro Glu Val 355 360 365 Gly Met Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu 370 375 380 His Asp Thr Thr Glu Tyr Lys Leu Arg Thr Leu Gln Val Ser Pro Leu 385 390 395 400 Asp Asn Gly Asp Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser 405 410 415 Trp Pro Asp His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe 420 425 430 Leu Asp Gln Ile Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly Pro 435 440 445 Ile Ile Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile 450 455 460 Val Ile Asp Met Leu Met Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys 465 470 475 480 Asp Ile Asp Ile Gln Lys Thr Ile Gln Met Val Arg Ala Gln Arg Ser 485 490 495 Gly Met Val Gln Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile 500 505 510 Ala Gln Phe Ile Glu Thr Thr Lys Lys Lys Leu Glu Val Leu Gln Ser 515 520 525 Gln Lys Gly Gln Glu Ser Glu Tyr Gly Asn Ile Thr Tyr Pro Pro Ala 530 535 540 Met Lys Asn Ala His Ala Lys Ala Ser Arg Thr Ser Ser Lys His Lys 545 550 555 560 Glu Asp Val Tyr Glu Asn Leu His Thr Lys Asn Lys Arg Glu Glu Lys 565 570 575 Val Lys Lys Gln Arg Ser Ala Asp Lys Glu Lys Ser Lys Gly Ser Leu 580 585 590 Lys Arg Lys 595 6214PRTArtificial SequencePTPN6 SH2 complete domain 6Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45 Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His 100 105 110 Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly 115 120 125 Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135 140 Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 145 150 155 160 Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175 Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180 185 190 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr 195 200 205 Leu Arg Gln Pro Tyr Tyr 210 797PRTArtificial SequencePTPN6 SH2 1 7Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr Leu Leu Lys 1 5 10 15 Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser Arg Lys Asn 20 25 30 Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln Val Thr His 35 40 45 Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr Gln Gln Gln 65 70 75 80 Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu Lys Tyr Pro 85 90 95 Leu 8104PRTArtificial SequencePTPN6 SH2 2 8Trp Tyr His Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln 1 5 10 15 Ala Lys Gly Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln 20 25 30 Pro Gly Asp Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly 35 40 45 Pro Gly Ser Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly 50 55 60 Gly Arg Tyr Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp 65 70 75 80 Leu Val Glu His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala 85 90 95 Phe Val Tyr Leu Arg Gln Pro Tyr 100 9597PRTHomo sapiens 9Met Thr Ser Arg Arg Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala 1 5 10 15 Glu Asn Leu Leu Leu Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg 20 25 30 Pro Ser Lys Ser Asn Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn 35 40 45 Gly Ala Val Thr His Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp 50 55 60 Leu Tyr Gly Gly Glu Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr 65 70 75 80 Tyr Met Glu His His Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile 85 90 95 Glu Leu Lys Tyr Pro Leu Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp 100 105 110 Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr Glu 115 120 125 Lys Gly Lys His Gly Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro 130 135 140 Gly Asp Phe Val Leu Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser 145 150 155 160 Asn Asp Gly Lys Ser Lys Val Thr His Val Met Ile Arg Cys Gln Glu 165

170 175 Leu Lys Tyr Asp Val Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp 180 185 190 Leu Val Glu His Tyr Lys Lys Asn Pro Met Val Glu Thr Leu Gly Thr 195 200 205 Val Leu Gln Leu Lys Gln Pro Leu Asn Thr Thr Arg Ile Asn Ala Ala 210 215 220 Glu Ile Glu Ser Arg Val Arg Glu Leu Ser Lys Leu Ala Glu Thr Thr 225 230 235 240 Asp Lys Val Lys Gln Gly Phe Trp Glu Glu Phe Glu Thr Leu Gln Gln 245 250 255 Gln Glu Cys Lys Leu Leu Tyr Ser Arg Lys Glu Gly Gln Arg Gln Glu 260 265 270 Asn Lys Asn Lys Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Thr 275 280 285 Arg Val Val Leu His Asp Gly Asp Pro Asn Glu Pro Val Ser Asp Tyr 290 295 300 Ile Asn Ala Asn Ile Ile Met Pro Glu Phe Glu Thr Lys Cys Asn Asn 305 310 315 320 Ser Lys Pro Lys Lys Ser Tyr Ile Ala Thr Gln Gly Cys Leu Gln Asn 325 330 335 Thr Val Asn Asp Phe Trp Arg Met Val Phe Gln Glu Asn Ser Arg Val 340 345 350 Ile Val Met Thr Thr Lys Glu Val Glu Arg Gly Lys Ser Lys Cys Val 355 360 365 Lys Tyr Trp Pro Asp Glu Tyr Ala Leu Lys Glu Tyr Gly Val Met Arg 370 375 380 Val Arg Asn Val Lys Glu Ser Ala Ala His Asp Tyr Thr Leu Arg Glu 385 390 395 400 Leu Lys Leu Ser Lys Val Gly Gln Ala Leu Leu Gln Gly Asn Thr Glu 405 410 415 Arg Thr Val Trp Gln Tyr His Phe Arg Thr Trp Pro Asp His Gly Val 420 425 430 Pro Ser Asp Pro Gly Gly Val Leu Asp Phe Leu Glu Glu Val His His 435 440 445 Lys Gln Glu Ser Ile Val Asp Ala Gly Pro Val Val Val His Cys Ser 450 455 460 Ala Gly Ile Gly Arg Thr Gly Thr Phe Ile Val Ile Asp Ile Leu Ile 465 470 475 480 Asp Ile Ile Arg Glu Lys Gly Val Asp Cys Asp Ile Asp Val Pro Lys 485 490 495 Thr Ile Gln Met Val Arg Ser Gln Arg Ser Gly Met Val Gln Thr Glu 500 505 510 Ala Gln Tyr Arg Phe Ile Tyr Met Ala Val Gln His Tyr Ile Glu Thr 515 520 525 Leu Gln Arg Arg Ile Glu Glu Glu Gln Lys Ser Lys Arg Lys Gly His 530 535 540 Glu Tyr Thr Asn Ile Lys Tyr Ser Leu Val Asp Gln Thr Ser Gly Asp 545 550 555 560 Gln Ser Pro Leu Pro Pro Cys Thr Pro Thr Pro Pro Cys Ala Glu Met 565 570 575 Arg Glu Asp Ser Ala Arg Val Tyr Glu Asn Val Gly Leu Met Gln Gln 580 585 590 Gln Arg Ser Phe Arg 595 1097PRTArtificial SequenceSHP-2 first SH2 domain 10Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu 11105PRTArtificial SequenceSHP-2 second SH2 domain 11Trp Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr 1 5 10 15 Glu Lys Gly Lys His Gly Ser Phe Leu Val Arg Glu Ser Gln Ser His 20 25 30 Pro Gly Asp Phe Val Leu Ser Val Arg Thr Gly Asp Asp Lys Gly Glu 35 40 45 Ser Asn Asp Gly Lys Ser Lys Val Thr His Val Met Ile Arg Cys Gln 50 55 60 Glu Leu Lys Tyr Asp Val Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr 65 70 75 80 Asp Leu Val Glu His Tyr Lys Lys Asn Pro Met Val Glu Thr Leu Gly 85 90 95 Thr Val Leu Gln Leu Lys Gln Pro Leu 100 105 12211PRTArtificial SequenceSHP-2 both SH2 domains 12Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp Phe His Gly His Leu 100 105 110 Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr Glu Lys Gly Lys His Gly 115 120 125 Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro Gly Asp Phe Val Leu 130 135 140 Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser Asn Asp Gly Lys Ser 145 150 155 160 Lys Val Thr His Val Met Ile Arg Cys Gln Glu Leu Lys Tyr Asp Val 165 170 175 Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp Leu Val Glu His Tyr 180 185 190 Lys Lys Asn Pro Met Val Glu Thr Leu Gly Thr Val Leu Gln Leu Lys 195 200 205 Gln Pro Leu 210 13112PRTArtificial SequenceCD3-zeta endodomain 13Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 14754PRTArtificial SequenceZAP70-SH2 domain fused to a CD3-zeta endodomain 14Met Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 1 5 10 15 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 20 25 30 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 35 40 45 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 50 55 60 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 65 70 75 80 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 85 90 95 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 100 105 110 Pro Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Met Pro Asp Pro Ala Ala His Leu Pro 130 135 140 Phe Phe Tyr Gly Ser Ile Ser Arg Ala Glu Ala Glu Glu His Leu Lys 145 150 155 160 Leu Ala Gly Met Ala Asp Gly Leu Phe Leu Leu Arg Gln Cys Leu Arg 165 170 175 Ser Leu Gly Gly Tyr Val Leu Ser Leu Val His Asp Val Arg Phe His 180 185 190 His Phe Pro Ile Glu Arg Gln Leu Asn Gly Thr Tyr Ala Ile Ala Gly 195 200 205 Gly Lys Ala His Cys Gly Pro Ala Glu Leu Cys Glu Phe Tyr Ser Arg 210 215 220 Asp Pro Asp Gly Leu Pro Cys Asn Leu Arg Lys Pro Cys Asn Arg Pro 225 230 235 240 Ser Gly Leu Glu Pro Gln Pro Gly Val Phe Asp Cys Leu Arg Asp Ala 245 250 255 Met Val Arg Asp Tyr Val Arg Gln Thr Trp Lys Leu Glu Gly Glu Ala 260 265 270 Leu Glu Gln Ala Ile Ile Ser Gln Ala Pro Gln Val Glu Lys Leu Ile 275 280 285 Ala Thr Thr Ala His Glu Arg Met Pro Trp Tyr His Ser Ser Leu Thr 290 295 300 Arg Glu Glu Ala Glu Arg Lys Leu Tyr Ser Gly Ala Gln Thr Asp Gly 305 310 315 320 Lys Phe Leu Leu Arg Pro Arg Lys Glu Gln Gly Thr Tyr Ala Leu Ser 325 330 335 Leu Ile Tyr Gly Lys Thr Val Tyr His Tyr Leu Ile Ser Gln Asp Lys 340 345 350 Ala Gly Lys Tyr Cys Ile Pro Glu Gly Thr Lys Phe Asp Thr Leu Trp 355 360 365 Gln Leu Val Glu Tyr Leu Lys Leu Lys Ala Asp Gly Leu Ile Tyr Cys 370 375 380 Leu Lys Glu Ala Cys Pro Asn Ser Ser Ala Ser Asn Ala Ser Gly Ala 385 390 395 400 Ala Ala Pro Thr Leu Pro Ala His Pro Ser Thr Leu Thr His Pro Gln 405 410 415 Arg Arg Ile Asp Thr Leu Asn Ser Asp Gly Tyr Thr Pro Glu Pro Ala 420 425 430 Arg Ile Thr Ser Pro Asp Lys Pro Arg Pro Met Pro Met Asp Thr Ser 435 440 445 Val Tyr Glu Ser Pro Tyr Ser Asp Pro Glu Glu Leu Lys Asp Lys Lys 450 455 460 Leu Phe Leu Lys Arg Asp Asn Leu Leu Ile Ala Asp Ile Glu Leu Gly 465 470 475 480 Cys Gly Asn Phe Gly Ser Val Arg Gln Gly Val Tyr Arg Met Arg Lys 485 490 495 Lys Gln Ile Asp Val Ala Ile Lys Val Leu Lys Gln Gly Thr Glu Lys 500 505 510 Ala Asp Thr Glu Glu Met Met Arg Glu Ala Gln Ile Met His Gln Leu 515 520 525 Asp Asn Pro Tyr Ile Val Arg Leu Ile Gly Val Cys Gln Ala Glu Ala 530 535 540 Leu Met Leu Val Met Glu Met Ala Gly Gly Gly Pro Leu His Lys Phe 545 550 555 560 Leu Val Gly Lys Arg Glu Glu Ile Pro Val Ser Asn Val Ala Glu Leu 565 570 575 Leu His Gln Val Ser Met Gly Met Lys Tyr Leu Glu Glu Lys Asn Phe 580 585 590 Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Val Asn Arg His 595 600 605 Tyr Ala Lys Ile Ser Asp Phe Gly Leu Ser Lys Ala Leu Gly Ala Asp 610 615 620 Asp Ser Tyr Tyr Thr Ala Arg Ser Ala Gly Lys Trp Pro Leu Lys Trp 625 630 635 640 Tyr Ala Pro Glu Cys Ile Asn Phe Arg Lys Phe Ser Ser Arg Ser Asp 645 650 655 Val Trp Ser Tyr Gly Val Thr Met Trp Glu Ala Leu Ser Tyr Gly Gln 660 665 670 Lys Pro Tyr Lys Lys Met Lys Gly Pro Glu Val Met Ala Phe Ile Glu 675 680 685 Gln Gly Lys Arg Met Glu Cys Pro Pro Glu Cys Pro Pro Glu Leu Tyr 690 695 700 Ala Leu Met Ser Asp Cys Trp Ile Tyr Lys Trp Glu Asp Arg Pro Asp 705 710 715 720 Phe Leu Thr Val Glu Gln Arg Met Arg Ala Cys Tyr Tyr Ser Leu Ala 725 730 735 Ser Lys Val Glu Gly Pro Pro Gly Ser Thr Gln Lys Ala Glu Ala Ala 740 745 750 Cys Ala 1597PRTArtificial SequencePDCD1 endodomain 15Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln 1 5 10 15 Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr 20 25 30 Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val 35 40 45 Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser 50 55 60 Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro 65 70 75 80 Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro 85 90 95 Leu 16141PRTArtificial SequenceBTLA4 endodomain 16Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly Leu Gln 1 5 10 15 Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn Lys Lys Val Arg 20 25 30 Arg Ala Pro Leu Ser Glu Gly Pro His Ser Leu Gly Cys Tyr Asn Pro 35 40 45 Met Met Glu Asp Gly Ile Ser Tyr Thr Thr Leu Arg Phe Pro Glu Met 50 55 60 Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu Met Gln Arg Pro 65 70 75 80 Pro Pro Asp Cys Asp Asp Thr Val Thr Tyr Ser Ala Leu His Lys Arg 85 90 95 Gln Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu Asp Glu 100 105 110 Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu Arg Pro 115 120 125 Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His 130 135 140 17168PRTArtificial SequenceLILRB1 endodomain 17Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys 1 5 10 15 Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp 20 25 30 Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu 35 40 45 Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu 50 55 60 Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 65 70 75 80 Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro 85 90 95 Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu 100 105 110 Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln 115 120 125 Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala 130 135 140 Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser 145 150 155 160 Ile Tyr Ala Thr Leu Ala Ile His 165 18101PRTArtificial SequenceLAIR1 endodomain 18His Arg Gln Asn Gln Ile Lys Gln Gly Pro Pro Arg Ser Lys Asp Glu 1 5 10 15 Glu Gln Lys Pro Gln Gln Arg Pro Asp Leu Ala Val Asp Val Leu Glu 20 25 30 Arg Thr Ala Asp Lys Ala Thr Val Asn Gly Leu Pro Glu Lys Asp Arg 35 40 45 Glu Thr Asp Thr Ser Ala Leu Ala Ala Gly Ser Ser Gln Glu Val Thr 50 55 60 Tyr Ala Gln Leu Asp His Trp Ala Leu Thr Gln Arg Thr Ala Arg Ala 65 70 75 80 Val Ser Pro Gln Ser Thr Lys Pro Met Ala Glu Ser Ile Thr Tyr Ala 85 90 95 Ala Val Ala Arg His 100 1962PRTArtificial SequenceCTLA4 endodomain 19Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe Tyr 1 5 10 15 Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys Arg

20 25 30 Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro 35 40 45 Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn 50 55 60 20111PRTArtificial SequenceKIR2DL1 endodomain 20Gly Asn Ser Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Ile 1 5 10 15 Ile Pro Phe Ala Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ala 20 25 30 Asn Lys Lys Asn Ala Val Val Met Asp Gln Glu Pro Ala Gly Asn Arg 35 40 45 Thr Val Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr 50 55 60 Tyr Thr Gln Leu Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg 65 70 75 80 Pro Ser Gln Arg Pro Lys Thr Pro Pro Thr Asp Ile Ile Val Tyr Thr 85 90 95 Glu Leu Pro Asn Ala Glu Ser Arg Ser Lys Val Val Ser Cys Pro 100 105 110 21143PRTArtificial SequenceKIR2DL4 endodomain 21Gly Ile Ala Arg His Leu His Ala Val Ile Arg Tyr Ser Val Ala Ile 1 5 10 15 Ile Leu Phe Thr Ile Leu Pro Phe Phe Leu Leu His Arg Trp Cys Ser 20 25 30 Lys Lys Lys Glu Asn Ala Ala Val Met Asn Gln Glu Pro Ala Gly His 35 40 45 Arg Thr Val Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val 50 55 60 Thr Tyr Ala Gln Leu Asp His Cys Ile Phe Thr Gln Arg Lys Ile Thr 65 70 75 80 Gly Pro Ser Gln Arg Ser Lys Arg Pro Ser Thr Asp Thr Ser Val Cys 85 90 95 Ile Glu Leu Pro Asn Ala Glu Pro Arg Ala Leu Ser Pro Ala His Glu 100 105 110 His His Ser Gln Ala Leu Met Gly Ser Ser Arg Glu Thr Thr Ala Leu 115 120 125 Ser Gln Thr Gln Leu Ala Ser Ser Asn Val Pro Ala Ala Gly Ile 130 135 140 22143PRTArtificial SequenceKIR2DL5 endodomain 22Thr Gly Ile Arg Arg His Leu His Ile Leu Ile Gly Thr Ser Val Ala 1 5 10 15 Ile Ile Leu Phe Ile Ile Leu Phe Phe Phe Leu Leu His Cys Cys Cys 20 25 30 Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asp 35 40 45 Arg Thr Val Asn Arg Glu Asp Ser Asp Asp Gln Asp Pro Gln Glu Val 50 55 60 Thr Tyr Ala Gln Leu Asp His Cys Val Phe Thr Gln Thr Lys Ile Thr 65 70 75 80 Ser Pro Ser Gln Arg Pro Lys Thr Pro Pro Thr Asp Thr Thr Met Tyr 85 90 95 Met Glu Leu Pro Asn Ala Lys Pro Arg Ser Leu Ser Pro Ala His Lys 100 105 110 His His Ser Gln Ala Leu Arg Gly Ser Ser Arg Glu Thr Thr Ala Leu 115 120 125 Ser Gln Asn Arg Val Ala Ser Ser His Val Pro Ala Ala Gly Ile 130 135 140 23111PRTArtificial SequenceKIR3DL1 endodomain 23Lys Asp Pro Arg His Leu His Ile Leu Ile Gly Thr Ser Val Val Ile 1 5 10 15 Ile Leu Phe Ile Leu Leu Leu Phe Phe Leu Leu His Leu Trp Cys Ser 20 25 30 Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asn Arg 35 40 45 Thr Ala Asn Ser Glu Asp Ser Asp Glu Gln Asp Pro Glu Glu Val Thr 50 55 60 Tyr Ala Gln Leu Asp His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg 65 70 75 80 Pro Ser Gln Arg Pro Lys Thr Pro Pro Thr Asp Thr Ile Leu Tyr Thr 85 90 95 Glu Leu Pro Asn Ala Lys Pro Arg Ser Lys Val Val Ser Cys Pro 100 105 110 2497PRTArtificial SequenceKIR3DL3 endodomain 24Lys Asp Pro Gly Asn Ser Arg His Leu His Val Leu Ile Gly Thr Ser 1 5 10 15 Val Val Ile Ile Pro Phe Ala Ile Leu Leu Phe Phe Leu Leu His Arg 20 25 30 Trp Cys Ala Asn Lys Lys Asn Ala Val Val Met Asp Gln Glu Pro Ala 35 40 45 Gly Asn Arg Thr Val Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro Gln 50 55 60 Glu Val Thr Tyr Ala Gln Leu Asn His Cys Val Phe Thr Gln Arg Lys 65 70 75 80 Ile Thr Arg Pro Ser Gln Arg Pro Lys Thr Pro Pro Thr Asp Thr Ser 85 90 95 Val 25701PRTArtificial SequencePTPN6-SH2 domain fused to a PD1 endodomain 25Met Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe 1 5 10 15 Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro 20 25 30 Glu Pro Pro Val Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile 35 40 45 Val Phe Pro Ser Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser 50 55 60 Ala Asp Gly Pro Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His 65 70 75 80 Cys Ser Trp Pro Leu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 85 90 95 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Val Arg Trp Phe His 100 105 110 Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr Leu Leu Lys Gly Arg Gly 115 120 125 Val His Gly Ser Phe Leu Ala Arg Pro Ser Arg Lys Asn Gln Gly Asp 130 135 140 Phe Ser Leu Ser Val Arg Val Gly Asp Gln Val Thr His Ile Arg Ile 145 150 155 160 Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr Gly Gly Glu Lys Phe Ala 165 170 175 Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr Gln Gln Gln Gly Val Leu 180 185 190 Gln Asp Arg Asp Gly Thr Ile Ile His Leu Lys Tyr Pro Leu Asn Cys 195 200 205 Ser Asp Pro Thr Ser Glu Arg Trp Tyr His Gly His Met Ser Gly Gly 210 215 220 Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly Glu Pro Trp Thr Phe Leu 225 230 235 240 Val Arg Glu Ser Leu Ser Gln Pro Gly Asp Phe Val Leu Ser Val Leu 245 250 255 Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser Pro Leu Arg Val Thr His 260 265 270 Ile Lys Val Met Cys Glu Gly Gly Arg Tyr Thr Val Gly Gly Leu Glu 275 280 285 Thr Phe Asp Ser Leu Thr Asp Leu Val Glu His Phe Lys Lys Thr Gly 290 295 300 Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr Leu Arg Gln Pro Tyr Tyr 305 310 315 320 Ala Thr Arg Val Asn Ala Ala Asp Ile Glu Asn Arg Val Leu Glu Leu 325 330 335 Asn Lys Lys Gln Glu Ser Glu Asp Thr Ala Lys Ala Gly Phe Trp Glu 340 345 350 Glu Phe Glu Ser Leu Gln Lys Gln Glu Val Lys Asn Leu His Gln Arg 355 360 365 Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly Lys Asn Arg Tyr Lys Asn 370 375 380 Ile Leu Pro Phe Asp His Ser Arg Val Ile Leu Gln Gly Arg Asp Ser 385 390 395 400 Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn Tyr Ile Lys Asn Gln 405 410 415 Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr Tyr Ile Ala Ser Gln Gly 420 425 430 Cys Leu Glu Ala Thr Val Asn Asp Phe Trp Gln Met Ala Trp Gln Glu 435 440 445 Asn Ser Arg Val Ile Val Met Thr Thr Arg Glu Val Glu Lys Gly Arg 450 455 460 Asn Lys Cys Val Pro Tyr Trp Pro Glu Val Gly Met Gln Arg Ala Tyr 465 470 475 480 Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu His Asp Thr Thr Glu Tyr 485 490 495 Lys Leu Arg Thr Leu Gln Val Ser Pro Leu Asp Asn Gly Asp Leu Ile 500 505 510 Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser Trp Pro Asp His Gly Val 515 520 525 Pro Ser Glu Pro Gly Gly Val Leu Ser Phe Leu Asp Gln Ile Asn Gln 530 535 540 Arg Gln Glu Ser Leu Pro His Ala Gly Pro Ile Ile Val His Cys Ser 545 550 555 560 Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile Val Ile Asp Met Leu Met 565 570 575 Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys Asp Ile Asp Ile Gln Lys 580 585 590 Thr Ile Gln Met Val Arg Ala Gln Arg Ser Gly Met Val Gln Thr Glu 595 600 605 Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile Ala Gln Phe Ile Glu Thr 610 615 620 Thr Lys Lys Lys Leu Glu Val Leu Gln Ser Gln Lys Gly Gln Glu Ser 625 630 635 640 Glu Tyr Gly Asn Ile Thr Tyr Pro Pro Ala Met Lys Asn Ala His Ala 645 650 655 Lys Ala Ser Arg Thr Ser Ser Lys His Lys Glu Asp Val Tyr Glu Asn 660 665 670 Leu His Thr Lys Asn Lys Arg Glu Glu Lys Val Lys Lys Gln Arg Ser 675 680 685 Ala Asp Lys Glu Lys Ser Lys Gly Ser Leu Lys Arg Lys 690 695 700 26299PRTArtificial SequenceZAP70 kinase domain 26Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn 1 5 10 15 Leu Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val 20 25 30 Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile 35 40 45 Lys Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met 50 55 60 Arg Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg 65 70 75 80 Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met 85 90 95 Ala Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu 100 105 110 Ile Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly 115 120 125 Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala 130 135 140 Arg Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe 145 150 155 160 Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg 165 170 175 Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn 180 185 190 Phe Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr 195 200 205 Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys 210 215 220 Gly Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys 225 230 235 240 Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp 245 250 255 Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg 260 265 270 Met Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro 275 280 285 Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 290 295 27280PRTArtificial SequencePTPN6 phosphatase domain 27Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu Val Lys Asn Leu 1 5 10 15 His Gln Arg Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly Lys Asn Arg 20 25 30 Tyr Lys Asn Ile Leu Pro Phe Asp His Ser Arg Val Ile Leu Gln Gly 35 40 45 Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn Tyr Ile 50 55 60 Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr Tyr Ile Ala 65 70 75 80 Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp Gln Met Ala 85 90 95 Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg Glu Val Glu 100 105 110 Lys Gly Arg Asn Lys Cys Val Pro Tyr Trp Pro Glu Val Gly Met Gln 115 120 125 Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu His Asp Thr 130 135 140 Thr Glu Tyr Lys Leu Arg Thr Leu Gln Val Ser Pro Leu Asp Asn Gly 145 150 155 160 Asp Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser Trp Pro Asp 165 170 175 His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe Leu Asp Gln 180 185 190 Ile Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly Pro Ile Ile Val 195 200 205 His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile Val Ile Asp 210 215 220 Met Leu Met Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys Asp Ile Asp 225 230 235 240 Ile Gln Lys Thr Ile Gln Met Val Arg Ala Gln Arg Ser Gly Met Val 245 250 255 Gln Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile Ala Gln Phe 260 265 270 Ile Glu Thr Thr Lys Lys Lys Leu 275 280 28274PRTArtificial SequenceSHP-2 phosphatase domain 28Trp Glu Glu Phe Glu Thr Leu Gln Gln Gln Glu Cys Lys Leu Leu Tyr 1 5 10 15 Ser Arg Lys Glu Gly Gln Arg Gln Glu Asn Lys Asn Lys Asn Arg Tyr 20 25 30 Lys Asn Ile Leu Pro Phe Asp His Thr Arg Val Val Leu His Asp Gly 35 40 45 Asp Pro Asn Glu Pro Val Ser Asp Tyr Ile Asn Ala Asn Ile Ile Met 50 55 60 Pro Glu Phe Glu Thr Lys Cys Asn Asn Ser Lys Pro Lys Lys Ser Tyr 65 70 75 80 Ile Ala Thr Gln Gly Cys Leu Gln Asn Thr Val Asn Asp Phe Trp Arg 85 90 95 Met Val Phe Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Lys Glu 100 105 110 Val Glu Arg Gly Lys Ser Lys Cys Val Lys Tyr Trp Pro Asp Glu Tyr 115 120 125 Ala Leu Lys Glu Tyr Gly Val Met Arg Val Arg Asn Val Lys Glu Ser 130 135 140 Ala Ala His Asp Tyr Thr Leu Arg Glu Leu Lys Leu Ser Lys Val Gly 145 150 155 160 Gln Ala Leu Leu Gln Gly Asn Thr Glu Arg Thr Val Trp Gln Tyr His 165 170 175 Phe Arg Thr Trp Pro Asp His Gly Val Pro Ser Asp Pro Gly Gly Val 180 185 190 Leu Asp Phe Leu Glu Glu Val His His Lys Gln Glu Ser Ile Met Asp 195 200 205 Ala Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly 210 215 220 Thr Phe Ile Val Ile Asp Ile Leu Ile Asp Ile Ile Arg Glu Lys Gly 225 230 235 240 Val Asp Cys Asp Ile Asp Val Pro Lys Thr Ile Gln Met Val Arg Ser 245 250 255 Gln Arg Ser Gly Met Val Gln Thr Glu Ala Gln Tyr Arg Phe Ile Tyr 260 265 270 Met Ala 29519PRTArtificial SequencePTPN6 SH2 domain fusion ZAP70 kinase domain 29Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys

Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45 Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His 100 105 110 Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly 115 120 125 Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135 140 Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 145 150 155 160 Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175 Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180 185 190 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr 195 200 205 Leu Arg Gln Pro Tyr Tyr Ser Gly Gly Gly Gly Ser Asp Pro Glu Glu 210 215 220 Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn Leu Leu Ile Ala 225 230 235 240 Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val Arg Gln Gly Val 245 250 255 Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile Lys Val Leu Lys 260 265 270 Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met Arg Glu Ala Gln 275 280 285 Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg Leu Ile Gly Val 290 295 300 Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met Ala Gly Gly Gly 305 310 315 320 Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu Ile Pro Val Ser 325 330 335 Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly Met Lys Tyr Leu 340 345 350 Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val Leu 355 360 365 Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe Gly Leu Ser Lys 370 375 380 Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg Ser Ala Gly Lys 385 390 395 400 Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn Phe Arg Lys Phe 405 410 415 Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr Met Trp Glu Ala 420 425 430 Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys Gly Pro Glu Val 435 440 445 Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys Pro Pro Glu Cys 450 455 460 Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp Ile Tyr Lys Trp 465 470 475 480 Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg Met Arg Ala Cys 485 490 495 Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro Gly Ser Thr Gln 500 505 510 Lys Ala Glu Ala Ala Cys Ala 515 30581PRTArtificial SequenceZAP70 SH2 domain fusion PTPN6 phosphatase domain 30Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe Trp Glu 290 295 300 Glu Phe Glu Ser Leu Gln Lys Gln Glu Val Lys Asn Leu His Gln Arg 305 310 315 320 Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly Lys Asn Arg Tyr Lys Asn 325 330 335 Ile Leu Pro Phe Asp His Ser Arg Val Ile Leu Gln Gly Arg Asp Ser 340 345 350 Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn Tyr Ile Lys Asn Gln 355 360 365 Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr Tyr Ile Ala Ser Gln Gly 370 375 380 Cys Leu Glu Ala Thr Val Asn Asp Phe Trp Gln Met Ala Trp Gln Glu 385 390 395 400 Asn Ser Arg Val Ile Val Met Thr Thr Arg Glu Val Glu Lys Gly Arg 405 410 415 Asn Lys Cys Val Pro Tyr Trp Pro Glu Val Gly Met Gln Arg Ala Tyr 420 425 430 Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu His Asp Thr Thr Glu Tyr 435 440 445 Lys Leu Arg Thr Leu Gln Val Ser Pro Leu Asp Asn Gly Asp Leu Ile 450 455 460 Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser Trp Pro Asp His Gly Val 465 470 475 480 Pro Ser Glu Pro Gly Gly Val Leu Ser Phe Leu Asp Gln Ile Asn Gln 485 490 495 Arg Gln Glu Ser Leu Pro His Ala Gly Pro Ile Ile Val His Cys Ser 500 505 510 Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile Val Ile Asp Met Leu Met 515 520 525 Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys Asp Ile Asp Ile Gln Lys 530 535 540 Thr Ile Gln Met Val Arg Ala Gln Arg Ser Gly Met Val Gln Thr Glu 545 550 555 560 Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile Ala Gln Phe Ile Glu Thr 565 570 575 Thr Lys Lys Lys Leu 580 3142PRTArtificial SequenceCD28 endodomain 31Met Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met 1 5 10 15 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 20 25 30 Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 3243PRTArtificial Sequence41BB endodomain 32Met Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 20 25 30 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 3337PRTArtificial SequenceOX40 endodomain 33Met Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 1 5 10 15 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 20 25 30 Thr Leu Ala Lys Ile 35 3438PRTArtificial SequenceICOS endodomain 34Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn 1 5 10 15 Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg 20 25 30 Leu Thr Asp Val Thr Leu 35 3548PRTArtificial SequenceCD27 endodomain 35Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro 1 5 10 15 Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr 20 25 30 Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro 35 40 45 36111PRTArtificial SequenceBTLA endodomain 36Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg 1 5 10 15 Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr Glu Ala 20 25 30 Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly Ile Tyr 35 40 45 Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser Glu Val 50 55 60 Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val Tyr Ala 65 70 75 80 Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu Ala Arg Asn 85 90 95 Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg Ser 100 105 110 37188PRTArtificial SequenceCD30 endodomain 37His Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys 1 5 10 15 Tyr Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg 20 25 30 Pro Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu 35 40 45 Pro Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr 50 55 60 Cys His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp 65 70 75 80 Ala Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro 85 90 95 Arg Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile 100 105 110 Met Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro 115 120 125 Glu Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu 130 135 140 Leu Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro 145 150 155 160 Pro Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly 165 170 175 Lys Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys 180 185 3858PRTArtificial SequenceGITR endodomain 38Gln Leu Gly Leu His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro 1 5 10 15 Arg Glu Thr Gln Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala 20 25 30 Arg Ser Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu 35 40 45 Glu Lys Gly Arg Leu Gly Asp Leu Trp Val 50 55 3960PRTArtificial SequenceHVEM endodomain 39Cys Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val 1 5 10 15 Ser Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile 20 25 30 Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu 35 40 45 Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His 50 55 60 40682PRTArtificial SequenceCD28 endodomain fused to amino-terminus of full-length ZAP 40Met Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met 1 5 10 15 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 20 25 30 Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met 50 55 60 Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser Arg 65 70 75 80 Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly Leu 85 90 95 Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu Ser 100 105 110 Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln Leu 115 120 125 Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro Ala 130 135 140 Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys Asn 145 150 155 160 Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro Gly 165 170 175 Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg Gln 180 185 190 Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser Gln 195 200 205 Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg Met 210 215 220 Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys Leu 225 230 235 240 Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg Lys 245 250 255 Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val Tyr 260 265 270 His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro Glu 275 280 285 Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys Leu 290 295 300 Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn Ser 305 310 315 320 Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala His 325 330 335 Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn Ser 340 345 350 Asp Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys Pro 355 360 365 Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser Asp 370 375 380 Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn Leu 385 390 395 400 Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val Arg 405 410 415 Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile Lys 420 425 430 Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met Arg 435 440 445 Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg Leu 450 455 460 Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met Ala 465 470 475 480 Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu Ile 485 490 495 Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly Met 500 505 510 Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala Arg 515 520 525 Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser

Asp Phe Gly 530 535 540 Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg Ser 545 550 555 560 Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn Phe 565 570 575 Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr Met 580 585 590 Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys Gly 595 600 605 Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys Pro 610 615 620 Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp Ile 625 630 635 640 Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg Met 645 650 655 Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro Gly 660 665 670 Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 675 680 41683PRTArtificial Sequence41BB endodomain fused to amino-terminus of full-length ZAP 41Met Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 20 25 30 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ser Gly Gly Gly Gly 35 40 45 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 65 70 75 80 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 85 90 95 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 100 105 110 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 115 120 125 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 130 135 140 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 145 150 155 160 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 165 170 175 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 180 185 190 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 195 200 205 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 210 215 220 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 225 230 235 240 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 245 250 255 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 260 265 270 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 275 280 285 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 290 295 300 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 305 310 315 320 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 325 330 335 His Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn 340 345 350 Ser Asp Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys 355 360 365 Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser 370 375 380 Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn 385 390 395 400 Leu Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val 405 410 415 Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile 420 425 430 Lys Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met 435 440 445 Arg Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg 450 455 460 Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met 465 470 475 480 Ala Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu 485 490 495 Ile Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly 500 505 510 Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala 515 520 525 Arg Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe 530 535 540 Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg 545 550 555 560 Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn 565 570 575 Phe Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr 580 585 590 Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys 595 600 605 Gly Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys 610 615 620 Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp 625 630 635 640 Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg 645 650 655 Met Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro 660 665 670 Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 675 680 42677PRTArtificial SequenceOX40 endodomain fused to amino-terminus of full-length ZAP 42Met Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 1 5 10 15 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 20 25 30 Thr Leu Ala Lys Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Pro Asp Pro Ala Ala 50 55 60 His Leu Pro Phe Phe Tyr Gly Ser Ile Ser Arg Ala Glu Ala Glu Glu 65 70 75 80 His Leu Lys Leu Ala Gly Met Ala Asp Gly Leu Phe Leu Leu Arg Gln 85 90 95 Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu Ser Leu Val His Asp Val 100 105 110 Arg Phe His His Phe Pro Ile Glu Arg Gln Leu Asn Gly Thr Tyr Ala 115 120 125 Ile Ala Gly Gly Lys Ala His Cys Gly Pro Ala Glu Leu Cys Glu Phe 130 135 140 Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys Asn Leu Arg Lys Pro Cys 145 150 155 160 Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro Gly Val Phe Asp Cys Leu 165 170 175 Arg Asp Ala Met Val Arg Asp Tyr Val Arg Gln Thr Trp Lys Leu Glu 180 185 190 Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser Gln Ala Pro Gln Val Glu 195 200 205 Lys Leu Ile Ala Thr Thr Ala His Glu Arg Met Pro Trp Tyr His Ser 210 215 220 Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys Leu Tyr Ser Gly Ala Gln 225 230 235 240 Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg Lys Glu Gln Gly Thr Tyr 245 250 255 Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val Tyr His Tyr Leu Ile Ser 260 265 270 Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro Glu Gly Thr Lys Phe Asp 275 280 285 Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys Leu Lys Ala Asp Gly Leu 290 295 300 Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn Ser Ser Ala Ser Asn Ala 305 310 315 320 Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala His Pro Ser Thr Leu Thr 325 330 335 His Pro Gln Arg Arg Ile Asp Thr Leu Asn Ser Asp Gly Tyr Thr Pro 340 345 350 Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys Pro Arg Pro Met Pro Met 355 360 365 Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser Asp Pro Glu Glu Leu Lys 370 375 380 Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn Leu Leu Ile Ala Asp Ile 385 390 395 400 Glu Leu Gly Cys Gly Asn Phe Gly Ser Val Arg Gln Gly Val Tyr Arg 405 410 415 Met Arg Lys Lys Gln Ile Asp Val Ala Ile Lys Val Leu Lys Gln Gly 420 425 430 Thr Glu Lys Ala Asp Thr Glu Glu Met Met Arg Glu Ala Gln Ile Met 435 440 445 His Gln Leu Asp Asn Pro Tyr Ile Val Arg Leu Ile Gly Val Cys Gln 450 455 460 Ala Glu Ala Leu Met Leu Val Met Glu Met Ala Gly Gly Gly Pro Leu 465 470 475 480 His Lys Phe Leu Val Gly Lys Arg Glu Glu Ile Pro Val Ser Asn Val 485 490 495 Ala Glu Leu Leu His Gln Val Ser Met Gly Met Lys Tyr Leu Glu Glu 500 505 510 Lys Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Val 515 520 525 Asn Arg His Tyr Ala Lys Ile Ser Asp Phe Gly Leu Ser Lys Ala Leu 530 535 540 Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg Ser Ala Gly Lys Trp Pro 545 550 555 560 Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn Phe Arg Lys Phe Ser Ser 565 570 575 Arg Ser Asp Val Trp Ser Tyr Gly Val Thr Met Trp Glu Ala Leu Ser 580 585 590 Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys Gly Pro Glu Val Met Ala 595 600 605 Phe Ile Glu Gln Gly Lys Arg Met Glu Cys Pro Pro Glu Cys Pro Pro 610 615 620 Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp Ile Tyr Lys Trp Glu Asp 625 630 635 640 Arg Pro Asp Phe Leu Thr Val Glu Gln Arg Met Arg Ala Cys Tyr Tyr 645 650 655 Ser Leu Ala Ser Lys Val Glu Gly Pro Pro Gly Ser Thr Gln Lys Ala 660 665 670 Glu Ala Ala Cys Ala 675 43276PRTArtificial SequenceCD28 endodomain fused to the amino-terminus of PTPN6 SH2 domain 43Met Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met 1 5 10 15 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 20 25 30 Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met 50 55 60 Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr Leu 65 70 75 80 Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser Arg 85 90 95 Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln Val 100 105 110 Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr Gly 115 120 125 Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr Gln 130 135 140 Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu Lys 145 150 155 160 Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His Gly 165 170 175 His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly Glu 180 185 190 Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp Phe 195 200 205 Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser Pro 210 215 220 Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr Thr 225 230 235 240 Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu His 245 250 255 Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr Leu 260 265 270 Arg Gln Pro Tyr 275 44277PRTArtificial Sequence41BB endodomain fused to the amino-terminus of PTPN6 SH2 domain 44Met Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 20 25 30 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ser Gly Gly Gly Gly 35 40 45 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 65 70 75 80 Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 85 90 95 Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 100 105 110 Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 115 120 125 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr 130 135 140 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 145 150 155 160 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His 165 170 175 Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly 180 185 190 Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp 195 200 205 Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 210 215 220 Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 225 230 235 240 Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 245 250 255 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr 260 265 270 Leu Arg Gln Pro Tyr 275 45271PRTArtificial SequenceOX40 endodomain fused to the amino-terminus of PTPN6 SH2 domain 45Met Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 1 5 10 15 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 20 25 30 Thr Leu Ala Lys Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Val Arg Trp Phe His 50 55 60 Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr Leu Leu Lys Gly Arg Gly 65 70 75 80 Val His Gly Ser Phe Leu Ala Arg Pro Ser Arg Lys Asn Gln Gly Asp 85 90 95 Phe Ser Leu Ser Val Arg Val Gly Asp Gln Val Thr His Ile Arg Ile 100 105 110 Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr Gly Gly Glu Lys Phe Ala 115 120 125 Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr Gln Gln Gln Gly Val Leu 130 135 140 Gln Asp Arg Asp Gly Thr Ile Ile His Leu Lys Tyr Pro Leu Asn Cys 145 150 155 160 Ser Asp Pro Thr Ser Glu Arg Trp Tyr His Gly His Met Ser Gly Gly 165 170 175 Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly Glu Pro Trp Thr Phe Leu

180 185 190 Val Arg Glu Ser Leu Ser Gln Pro Gly Asp Phe Val Leu Ser Val Leu 195 200 205 Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser Pro Leu Arg Val Thr His 210 215 220 Ile Lys Val Met Cys Glu Gly Gly Arg Tyr Thr Val Gly Gly Leu Glu 225 230 235 240 Thr Phe Asp Ser Leu Thr Asp Leu Val Glu His Phe Lys Lys Thr Gly 245 250 255 Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr Leu Arg Gln Pro Tyr 260 265 270 46350PRTArtificial SequenceAkt kinase domain 46Ala Glu Glu Met Glu Val Ser Leu Ala Lys Pro Lys His Arg Val Thr 1 5 10 15 Met Asn Glu Phe Glu Tyr Leu Lys Leu Leu Gly Lys Gly Thr Phe Gly 20 25 30 Lys Val Ile Leu Val Lys Glu Lys Ala Thr Gly Arg Tyr Tyr Ala Met 35 40 45 Lys Ile Leu Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val Ala His 50 55 60 Thr Leu Thr Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro Phe Leu 65 70 75 80 Thr Ala Leu Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys Phe Val 85 90 95 Met Glu Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser Arg Glu 100 105 110 Arg Val Phe Ser Glu Asp Arg Ala Arg Phe Tyr Gly Ala Glu Ile Val 115 120 125 Ser Ala Leu Asp Tyr Leu His Ser Glu Lys Asn Val Val Tyr Arg Asp 130 135 140 Leu Lys Leu Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile Lys Ile 145 150 155 160 Thr Asp Phe Gly Leu Cys Lys Glu Gly Ile Lys Asp Gly Ala Thr Met 165 170 175 Lys Thr Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Leu Glu 180 185 190 Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly Val Val 195 200 205 Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln Asp His 210 215 220 Glu Lys Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg Phe Pro Arg 225 230 235 240 Thr Leu Gly Pro Glu Ala Lys Ser Leu Leu Ser Gly Leu Leu Lys Lys 245 250 255 Asp Pro Lys Gln Arg Leu Gly Gly Gly Ser Glu Asp Ala Lys Glu Ile 260 265 270 Met Gln His Arg Phe Phe Ala Gly Ile Val Trp Gln His Val Tyr Glu 275 280 285 Lys Lys Leu Ser Pro Pro Phe Lys Pro Gln Val Thr Ser Glu Thr Asp 290 295 300 Thr Arg Tyr Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr Ile Thr 305 310 315 320 Pro Pro Asp Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu Arg Arg 325 330 335 Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Gly Thr Ala 340 345 350 47630PRTArtificial SequenceZAP70-SH2 domain fused directly to an Akt kinase domain 47Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Ala Glu Glu Met Glu Val Ser Leu 275 280 285 Ala Lys Pro Lys His Arg Val Thr Met Asn Glu Phe Glu Tyr Leu Lys 290 295 300 Leu Leu Gly Lys Gly Thr Phe Gly Lys Val Ile Leu Val Lys Glu Lys 305 310 315 320 Ala Thr Gly Arg Tyr Tyr Ala Met Lys Ile Leu Lys Lys Glu Val Ile 325 330 335 Val Ala Lys Asp Glu Val Ala His Thr Leu Thr Glu Asn Arg Val Leu 340 345 350 Gln Asn Ser Arg His Pro Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln 355 360 365 Thr His Asp Arg Leu Cys Phe Val Met Glu Tyr Ala Asn Gly Gly Glu 370 375 380 Leu Phe Phe His Leu Ser Arg Glu Arg Val Phe Ser Glu Asp Arg Ala 385 390 395 400 Arg Phe Tyr Gly Ala Glu Ile Val Ser Ala Leu Asp Tyr Leu His Ser 405 410 415 Glu Lys Asn Val Val Tyr Arg Asp Leu Lys Leu Glu Asn Leu Met Leu 420 425 430 Asp Lys Asp Gly His Ile Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu 435 440 445 Gly Ile Lys Asp Gly Ala Thr Met Lys Thr Phe Cys Gly Thr Pro Glu 450 455 460 Tyr Leu Ala Pro Glu Val Leu Glu Asp Asn Asp Tyr Gly Arg Ala Val 465 470 475 480 Asp Trp Trp Gly Leu Gly Val Val Met Tyr Glu Met Met Cys Gly Arg 485 490 495 Leu Pro Phe Tyr Asn Gln Asp His Glu Lys Leu Phe Glu Leu Ile Leu 500 505 510 Met Glu Glu Ile Arg Phe Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser 515 520 525 Leu Leu Ser Gly Leu Leu Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly 530 535 540 Gly Ser Glu Asp Ala Lys Glu Ile Met Gln His Arg Phe Phe Ala Gly 545 550 555 560 Ile Val Trp Gln His Val Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys 565 570 575 Pro Gln Val Thr Ser Glu Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe 580 585 590 Thr Ala Gln Met Ile Thr Ile Thr Pro Pro Asp Gln Asp Asp Ser Met 595 600 605 Glu Cys Val Asp Ser Glu Arg Arg Pro His Phe Pro Gln Phe Ser Tyr 610 615 620 Ser Ala Ser Gly Thr Ala 625 630 48651PRTArtificial SequenceZAP70-SH2 domain fused to an Akt kinase domain via a linker 48Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Glu 290 295 300 Met Glu Val Ser Leu Ala Lys Pro Lys His Arg Val Thr Met Asn Glu 305 310 315 320 Phe Glu Tyr Leu Lys Leu Leu Gly Lys Gly Thr Phe Gly Lys Val Ile 325 330 335 Leu Val Lys Glu Lys Ala Thr Gly Arg Tyr Tyr Ala Met Lys Ile Leu 340 345 350 Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val Ala His Thr Leu Thr 355 360 365 Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro Phe Leu Thr Ala Leu 370 375 380 Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys Phe Val Met Glu Tyr 385 390 395 400 Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser Arg Glu Arg Val Phe 405 410 415 Ser Glu Asp Arg Ala Arg Phe Tyr Gly Ala Glu Ile Val Ser Ala Leu 420 425 430 Asp Tyr Leu His Ser Glu Lys Asn Val Val Tyr Arg Asp Leu Lys Leu 435 440 445 Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile Lys Ile Thr Asp Phe 450 455 460 Gly Leu Cys Lys Glu Gly Ile Lys Asp Gly Ala Thr Met Lys Thr Phe 465 470 475 480 Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Leu Glu Asp Asn Asp 485 490 495 Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly Val Val Met Tyr Glu 500 505 510 Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln Asp His Glu Lys Leu 515 520 525 Phe Glu Leu Ile Leu Met Glu Glu Ile Arg Phe Pro Arg Thr Leu Gly 530 535 540 Pro Glu Ala Lys Ser Leu Leu Ser Gly Leu Leu Lys Lys Asp Pro Lys 545 550 555 560 Gln Arg Leu Gly Gly Gly Ser Glu Asp Ala Lys Glu Ile Met Gln His 565 570 575 Arg Phe Phe Ala Gly Ile Val Trp Gln His Val Tyr Glu Lys Lys Leu 580 585 590 Ser Pro Pro Phe Lys Pro Gln Val Thr Ser Glu Thr Asp Thr Arg Tyr 595 600 605 Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr Ile Thr Pro Pro Asp 610 615 620 Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu Arg Arg Pro His Phe 625 630 635 640 Pro Gln Phe Ser Tyr Ser Ala Ser Gly Thr Ala 645 650 49630PRTArtificial SequenceZAP70 mutated to be non-functional and fused to an Akt kinase domain 49Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Lys Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Ala Glu Glu Met Glu Val Ser Leu 275 280 285 Ala Lys Pro Lys His Arg Val Thr Met Asn Glu Phe Glu Tyr Leu Lys 290 295 300 Leu Leu Gly Lys Gly Thr Phe Gly Lys Val Ile Leu Val Lys Glu Lys 305 310 315 320 Ala Thr Gly Arg Tyr Tyr Ala Met Lys Ile Leu Lys Lys Glu Val Ile 325 330 335 Val Ala Lys Asp Glu Val Ala His Thr Leu Thr Glu Asn Arg Val Leu 340 345 350 Gln Asn Ser Arg His Pro Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln 355 360 365 Thr His Asp Arg Leu Cys Phe Val Met Glu Tyr Ala Asn Gly Gly Glu 370 375 380 Leu Phe Phe His Leu Ser Arg Glu Arg Val Phe Ser Glu Asp Arg Ala 385 390 395 400 Arg Phe Tyr Gly Ala Glu Ile Val Ser Ala Leu Asp Tyr Leu His Ser 405 410 415 Glu Lys Asn Val Val Tyr Arg Asp Leu Lys Leu Glu Asn Leu Met Leu 420 425 430 Asp Lys Asp Gly His Ile Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu 435 440 445 Gly Ile Lys Asp Gly Ala Thr Met Lys Thr Phe Cys Gly Thr Pro Glu 450 455 460 Tyr Leu Ala Pro Glu Val Leu Glu Asp Asn Asp Tyr Gly Arg Ala Val 465 470 475 480 Asp Trp Trp Gly Leu Gly Val Val Met Tyr Glu Met Met Cys Gly Arg 485 490 495 Leu Pro Phe Tyr Asn Gln Asp His Glu Lys Leu Phe Glu Leu Ile Leu 500 505 510 Met Glu Glu Ile Arg Phe Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser 515 520 525 Leu Leu Ser Gly Leu Leu Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly 530 535 540

Gly Ser Glu Asp Ala Lys Glu Ile Met Gln His Arg Phe Phe Ala Gly 545 550 555 560 Ile Val Trp Gln His Val Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys 565 570 575 Pro Gln Val Thr Ser Glu Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe 580 585 590 Thr Ala Gln Met Ile Thr Ile Thr Pro Pro Asp Gln Asp Asp Ser Met 595 600 605 Glu Cys Val Asp Ser Glu Arg Arg Pro His Phe Pro Gln Phe Ser Tyr 610 615 620 Ser Ala Ser Gly Thr Ala 625 630 50590PRTArtificial SequenceKinase containing domain of JAK2 50Arg Asn Glu Asp Leu Ile Phe Asn Glu Ser Leu Gly Gln Gly Thr Phe 1 5 10 15 Thr Lys Ile Phe Lys Gly Val Arg Arg Glu Val Gly Asp Tyr Gly Gln 20 25 30 Leu His Glu Thr Glu Val Leu Leu Lys Val Leu Asp Lys Ala His Arg 35 40 45 Asn Tyr Ser Glu Ser Phe Phe Glu Ala Ala Ser Met Met Ser Lys Leu 50 55 60 Ser His Lys His Leu Val Leu Asn Tyr Gly Val Cys Val Cys Gly Asp 65 70 75 80 Glu Asn Ile Leu Val Gln Glu Phe Val Lys Phe Gly Ser Leu Asp Thr 85 90 95 Tyr Leu Lys Lys Asn Lys Asn Cys Ile Asn Ile Leu Trp Lys Leu Glu 100 105 110 Val Ala Lys Gln Leu Ala Trp Ala Met His Phe Leu Glu Glu Asn Thr 115 120 125 Leu Ile His Gly Asn Val Cys Ala Lys Asn Ile Leu Leu Ile Arg Glu 130 135 140 Glu Asp Arg Lys Thr Gly Asn Pro Pro Phe Ile Lys Leu Ser Asp Pro 145 150 155 160 Gly Ile Ser Ile Thr Val Leu Pro Lys Asp Ile Leu Gln Glu Arg Ile 165 170 175 Pro Trp Val Pro Pro Glu Cys Ile Glu Asn Pro Lys Asn Leu Asn Leu 180 185 190 Ala Thr Asp Lys Trp Ser Phe Gly Thr Thr Leu Trp Glu Ile Cys Ser 195 200 205 Gly Gly Asp Lys Pro Leu Ser Ala Leu Asp Ser Gln Arg Lys Leu Gln 210 215 220 Phe Tyr Glu Asp Arg His Gln Leu Pro Ala Pro Lys Trp Ala Glu Leu 225 230 235 240 Ala Asn Leu Ile Asn Asn Cys Met Asp Tyr Glu Pro Asp Phe Arg Pro 245 250 255 Ser Phe Arg Ala Ile Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp 260 265 270 Tyr Glu Leu Leu Thr Glu Asn Asp Met Leu Pro Asn Met Arg Ile Gly 275 280 285 Ala Leu Gly Phe Ser Gly Ala Phe Glu Asp Arg Asp Pro Thr Gln Phe 290 295 300 Glu Glu Arg His Leu Lys Phe Leu Gln Gln Leu Gly Lys Gly Asn Phe 305 310 315 320 Gly Ser Val Glu Met Cys Arg Tyr Asp Pro Leu Gln Asp Asn Thr Gly 325 330 335 Glu Val Val Ala Val Lys Lys Leu Gln His Ser Thr Glu Glu His Leu 340 345 350 Arg Asp Phe Glu Arg Glu Ile Glu Ile Leu Lys Ser Leu Gln His Asp 355 360 365 Asn Ile Val Lys Tyr Lys Gly Val Cys Tyr Ser Ala Gly Arg Arg Asn 370 375 380 Leu Lys Leu Ile Met Glu Tyr Leu Pro Tyr Gly Ser Leu Arg Asp Tyr 385 390 395 400 Leu Gln Lys His Lys Glu Arg Ile Asp His Ile Lys Leu Leu Gln Tyr 405 410 415 Thr Ser Gln Ile Cys Lys Gly Met Glu Tyr Leu Gly Thr Lys Arg Tyr 420 425 430 Ile His Arg Asp Leu Ala Thr Arg Asn Ile Leu Val Glu Asn Glu Asn 435 440 445 Arg Val Lys Ile Gly Asp Phe Gly Leu Thr Lys Val Leu Pro Gln Asp 450 455 460 Lys Glu Tyr Tyr Lys Val Lys Glu Pro Gly Glu Ser Pro Ile Phe Trp 465 470 475 480 Tyr Ala Pro Glu Ser Leu Thr Glu Ser Lys Phe Ser Val Ala Ser Asp 485 490 495 Val Trp Ser Phe Gly Val Val Leu Tyr Glu Leu Phe Thr Tyr Ile Glu 500 505 510 Lys Ser Lys Ser Pro Pro Ala Glu Phe Met Arg Met Ile Gly Asn Asp 515 520 525 Lys Gln Gly Gln Met Ile Val Phe His Leu Ile Glu Leu Leu Lys Asn 530 535 540 Asn Gly Arg Leu Pro Arg Pro Asp Gly Cys Pro Asp Glu Ile Tyr Met 545 550 555 560 Ile Met Thr Glu Cys Trp Asn Asn Asn Val Asn Gln Arg Pro Ser Phe 565 570 575 Arg Asp Leu Ala Leu Arg Val Asp Gln Ile Arg Asp Asn Met 580 585 590 517PRTArtificial Sequenceconsensus Tobacco Etch Virus (TEV) cleavage site 51Glu Asn Leu Tyr Phe Gln Ser 1 5 52240PRTArtificial SequenceTeV protease domain 52Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile 1 5 10 15 Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly 20 25 30 Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg 35 40 45 Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val 50 55 60 Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met 65 70 75 80 Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu 85 90 95 Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr 100 105 110 Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys 115 120 125 Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr 130 135 140 Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe 145 150 155 160 Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr 165 170 175 Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu 180 185 190 Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu 195 200 205 Trp Gly Gly His Lys Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gln 210 215 220 Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln 225 230 235 240 53541PRTArtificial SequenceZAP70-SH2 domain fused to a TEV protease sequence 53Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Leu Phe 290 295 300 Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu 305 310 315 320 Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe 325 330 335 Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly 340 345 350 Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr 355 360 365 Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile 370 375 380 Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg 385 390 395 400 Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln 405 410 415 Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro 420 425 430 Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly 435 440 445 Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly 450 455 460 Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser 465 470 475 480 Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln 485 490 495 Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly 500 505 510 His Lys Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gln Pro Val Lys 515 520 525 Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln 530 535 540 54460PRTArtificial SequencePTPN6-SH2 domain fused to a TEV protease sequence 54Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45 Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His 100 105 110 Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly 115 120 125 Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135 140 Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 145 150 155 160 Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175 Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180 185 190 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr 195 200 205 Leu Arg Gln Pro Tyr Tyr Ser Gly Gly Gly Gly Ser Ser Leu Phe Lys 210 215 220 Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr 225 230 235 240 Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly 245 250 255 Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr 260 265 270 Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr Thr 275 280 285 Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg 290 295 300 Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu 305 310 315 320 Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr 325 330 335 Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser 340 345 350 Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln 355 360 365 Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile 370 375 380 His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val 385 390 395 400 Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp 405 410 415 Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His 420 425 430 Lys Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu 435 440 445 Ala Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln 450 455 460 55438PRTArtificial Sequencemembrane tethered transcription factor 55Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Asp His Ala Asp Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly 20 25 30 Gly Gly Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser 35 40 45 Thr Asn Val Ser Pro Ala Lys Pro Thr Thr Thr Ala Cys Pro Tyr Ser 50 55 60 Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro 65 70 75 80 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 85 90 95 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 100 105 110 Asp Phe Ala Cys Asp Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 115 120 125 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg 130 135 140 His Arg Arg Arg Gln Ala Glu Arg Met Ala Gln Ile Lys Arg Val Val 145 150 155 160 Ser Glu Lys Lys Thr Ala Gln Ala Pro His Arg Phe Gln Lys Thr Cys 165 170 175 Ser Pro Ile Ser Gly Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Met 180 185 190 Pro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser Leu Gly 195 200 205 Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp 210 215 220 Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro 225 230 235 240 Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp 245 250 255 Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile 260 265 270 Asp Glu Tyr Gly Gly Ser Gly Gly Gly Ser Met Gln Ile Leu Val Ala 275 280 285 Ser Asp Ala Thr Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp 290 295 300 Ile Cys Arg Leu Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys 305 310 315 320 Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr

325 330 335 Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg 340 345 350 Leu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp 355 360 365 Leu Asp Met Ile Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu 370 375 380 Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr 385 390 395 400 Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln 405 410 415 His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly 420 425 430 Gln Arg Gln Leu Thr Val 435 56438PRTArtificial Sequencetarget receptor 56Met Ala Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp 35 40 45 Ile Tyr Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Lys 100 105 110 Asn Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly 145 150 155 160 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 165 170 175 Gly Phe Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro 180 185 190 Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly Gly Ser 195 200 205 Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 210 215 220 Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr Thr Gly Gly 245 250 255 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met 260 265 270 Asp Pro Ala Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val 275 280 285 His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro 290 295 300 Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr 305 310 315 320 Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val Trp 325 330 335 Val Leu Ala Val Ile Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala 340 345 350 Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val 355 360 365 Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr 370 375 380 Pro Glu Pro Pro Val Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr 385 390 395 400 Ile Val Phe Pro Ser Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly 405 410 415 Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly 420 425 430 His Cys Ser Trp Pro Leu 435 57509PRTArtificial Sequencereceptor containing a CAR against CD19 with a cleavable CD3-zeta endodomain 57Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly 145 150 155 160 Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly 165 170 175 Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg 180 185 190 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr 195 200 205 Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser 210 215 220 Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr 225 230 235 240 Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295 300 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Phe Trp 305 310 315 320 Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 325 330 335 Thr Val Ala Phe Ile Ile Phe Trp Val Arg Cys Arg His Arg Arg Arg 340 345 350 Gln Ala Glu Arg Met Ala Gln Ile Lys Arg Val Val Ser Glu Lys Lys 355 360 365 Thr Ala Gln Ala Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile Ser 370 375 380 Gly Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Met Arg Arg Val Lys 385 390 395 400 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 405 410 415 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 420 425 430 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 435 440 445 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 450 455 460 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 465 470 475 480 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 485 490 495 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 58827PRTArtificial Sequencereceptor containing a CAR against CD19 with a CD3-zeta endodomain and a cleavable CD148 endodomain 58Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly 145 150 155 160 Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly 165 170 175 Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg 180 185 190 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr 195 200 205 Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser 210 215 220 Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr 225 230 235 240 Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295 300 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Phe Trp 305 310 315 320 Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 325 330 335 Thr Val Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg 340 345 350 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 385 390 395 400 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 405 410 415 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 420 425 430 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445 Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Asn Leu Tyr Phe Gln 450 455 460 Met Ala Val Phe Gly Cys Ile Phe Gly Ala Leu Val Ile Val Thr Val 465 470 475 480 Gly Gly Phe Ile Phe Trp Arg Lys Lys Arg Lys Asp Ala Lys Asn Asn 485 490 495 Glu Val Ser Phe Ser Gln Ile Lys Pro Lys Lys Ser Lys Leu Ile Arg 500 505 510 Val Glu Asn Phe Glu Ala Tyr Phe Lys Lys Gln Gln Ala Asp Ser Asn 515 520 525 Cys Gly Phe Ala Glu Glu Tyr Glu Asp Leu Lys Leu Val Gly Ile Ser 530 535 540 Gln Pro Lys Tyr Ala Ala Glu Leu Ala Glu Asn Arg Gly Lys Asn Arg 545 550 555 560 Tyr Asn Asn Val Leu Pro Tyr Asp Ile Ser Arg Val Lys Leu Ser Val 565 570 575 Gln Thr His Ser Thr Asp Asp Tyr Ile Asn Ala Asn Tyr Met Pro Gly 580 585 590 Tyr His Ser Lys Lys Asp Phe Ile Ala Thr Gln Gly Pro Leu Pro Asn 595 600 605 Thr Leu Lys Asp Phe Trp Arg Met Val Trp Glu Lys Asn Val Tyr Ala 610 615 620 Ile Ile Met Leu Thr Lys Cys Val Glu Gln Gly Arg Thr Lys Cys Glu 625 630 635 640 Glu Tyr Trp Pro Ser Lys Gln Ala Gln Asp Tyr Gly Asp Ile Thr Val 645 650 655 Ala Met Thr Ser Glu Ile Val Leu Pro Glu Trp Thr Ile Arg Asp Phe 660 665 670 Thr Val Lys Asn Ile Gln Thr Ser Glu Ser His Pro Leu Arg Gln Phe 675 680 685 His Phe Thr Ser Trp Pro Asp His Gly Val Pro Asp Thr Thr Asp Leu 690 695 700 Leu Ile Asn Phe Arg Tyr Leu Val Arg Asp Tyr Met Lys Gln Ser Pro 705 710 715 720 Pro Glu Ser Pro Ile Leu Val His Cys Ser Ala Gly Val Gly Arg Thr 725 730 735 Gly Thr Phe Ile Ala Ile Asp Arg Leu Ile Tyr Gln Ile Glu Asn Glu 740 745 750 Asn Thr Val Asp Val Tyr Gly Ile Val Tyr Asp Leu Arg Met His Arg 755 760 765 Pro Leu Met Val Gln Thr Glu Asp Gln Tyr Val Phe Leu Asn Gln Cys 770 775 780 Val Leu Asp Ile Val Arg Ser Gln Lys Asp Ser Lys Val Asp Leu Ile 785 790 795 800 Tyr Gln Asn Thr Thr Ala Met Thr Ile Tyr Glu Asn Leu Ala Pro Val 805 810 815 Thr Thr Phe Gly Lys Thr Asn Gly Tyr Ile Ala 820 825 5920PRTArtificial Sequence2a self-cleaving peptide 59Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu 1 5 10 15 Asn Pro Gly Pro 20 6020PRTArtificial Sequence2a self-cleaving peptide 60Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 61563PRTArtificial Sequencedual SH2 domains from SHP-2 fused to ZAP70 kinase domain 61Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp Phe His Gly His Leu 100 105 110 Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr Glu Lys Gly Lys His Gly 115 120 125 Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro Gly Asp Phe Val Leu 130 135 140 Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser Asn Asp Gly Lys Ser 145 150 155 160 Lys Val Thr His Val Met Ile Arg Cys Gln Glu Leu Lys Tyr Asp Val 165 170 175 Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp Leu Val Glu His Tyr 180 185 190 Lys Lys Asn Pro Met Val Glu Thr Leu Gly Thr Val Leu Gln Leu Lys 195 200 205 Gln Pro Leu Asn Thr Thr Arg Ile Asn Pro Asn Ser Ser Ala Ser Asn 210 215 220 Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala His Pro Ser Thr Leu 225 230 235 240 Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn Ser Asp Gly Tyr Thr 245 250 255 Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys Pro Arg Pro Met Pro 260 265 270 Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser Asp Pro Glu Glu Leu 275 280 285 Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn Leu Leu Ile Ala Asp 290 295 300 Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val Arg Gln Gly Val Tyr 305 310 315

320 Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile Lys Val Leu Lys Gln 325 330 335 Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met Arg Glu Ala Gln Ile 340 345 350 Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg Leu Ile Gly Val Cys 355 360 365 Gln Ala Glu Ala Leu Met Leu Val Met Glu Met Ala Gly Gly Gly Pro 370 375 380 Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu Ile Pro Val Ser Asn 385 390 395 400 Val Ala Glu Leu Leu His Gln Val Ser Met Gly Met Lys Tyr Leu Glu 405 410 415 Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Leu 420 425 430 Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe Gly Leu Ser Lys Ala 435 440 445 Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg Ser Ala Gly Lys Trp 450 455 460 Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn Phe Arg Lys Phe Ser 465 470 475 480 Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr Met Trp Glu Ala Leu 485 490 495 Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys Gly Pro Glu Val Met 500 505 510 Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys Pro Pro Glu Cys Pro 515 520 525 Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp Ile Tyr Lys Trp Glu 530 535 540 Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg Met Arg Ala Cys Tyr 545 550 555 560 Tyr Ser Leu 62567PRTArtificial Sequencedual SHP-2 SH2 domain fused to an Akt kinase domain 62Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp Phe His Gly His Leu 100 105 110 Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr Glu Lys Gly Lys His Gly 115 120 125 Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro Gly Asp Phe Val Leu 130 135 140 Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser Asn Asp Gly Lys Ser 145 150 155 160 Lys Val Thr His Val Met Ile Arg Cys Gln Glu Leu Lys Tyr Asp Val 165 170 175 Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp Leu Val Glu His Tyr 180 185 190 Lys Lys Asn Pro Met Val Glu Thr Leu Gly Thr Val Leu Gln Leu Lys 195 200 205 Gln Pro Leu Asn Thr Thr Arg Ile Asn Ala Glu Glu Met Glu Val Ser 210 215 220 Leu Ala Lys Pro Lys His Arg Val Thr Met Asn Glu Phe Glu Tyr Leu 225 230 235 240 Lys Leu Leu Gly Lys Gly Thr Phe Gly Lys Val Ile Leu Val Lys Glu 245 250 255 Lys Ala Thr Gly Arg Tyr Tyr Ala Met Lys Ile Leu Lys Lys Glu Val 260 265 270 Ile Val Ala Lys Asp Glu Val Ala His Thr Leu Thr Glu Asn Arg Val 275 280 285 Leu Gln Asn Ser Arg His Pro Phe Leu Thr Ala Leu Lys Tyr Ser Phe 290 295 300 Gln Thr His Asp Arg Leu Cys Phe Val Met Glu Tyr Ala Asn Gly Gly 305 310 315 320 Glu Leu Phe Phe His Leu Ser Arg Glu Arg Val Phe Ser Glu Asp Arg 325 330 335 Ala Arg Phe Tyr Gly Ala Glu Ile Val Ser Ala Leu Asp Tyr Leu His 340 345 350 Ser Glu Lys Asn Val Val Tyr Arg Asp Leu Lys Leu Glu Asn Leu Met 355 360 365 Leu Asp Lys Asp Gly His Ile Lys Ile Thr Asp Phe Gly Leu Cys Lys 370 375 380 Glu Gly Ile Lys Asp Gly Ala Thr Met Lys Thr Phe Cys Gly Thr Pro 385 390 395 400 Glu Tyr Leu Ala Pro Glu Val Leu Glu Asp Asn Asp Tyr Gly Arg Ala 405 410 415 Val Asp Trp Trp Gly Leu Gly Val Val Met Tyr Glu Met Met Cys Gly 420 425 430 Arg Leu Pro Phe Tyr Asn Gln Asp His Glu Lys Leu Phe Glu Leu Ile 435 440 445 Leu Met Glu Glu Ile Arg Phe Pro Arg Thr Leu Gly Pro Glu Ala Lys 450 455 460 Ser Leu Leu Ser Gly Leu Leu Lys Lys Asp Pro Lys Gln Arg Leu Gly 465 470 475 480 Gly Gly Ser Glu Asp Ala Lys Glu Ile Met Gln His Arg Phe Phe Ala 485 490 495 Gly Ile Val Trp Gln His Val Tyr Glu Lys Lys Leu Ser Pro Pro Phe 500 505 510 Lys Pro Gln Val Thr Ser Glu Thr Asp Thr Arg Tyr Phe Asp Glu Glu 515 520 525 Phe Thr Ala Gln Met Ile Thr Ile Thr Pro Pro Asp Gln Asp Asp Ser 530 535 540 Met Glu Cys Val Asp Ser Glu Arg Arg Pro His Phe Pro Gln Phe Ser 545 550 555 560 Tyr Ser Ala Ser Gly Thr Ala 565 636PRTArtificial SequenceITIM conserved sequenceMISC_FEATURE(1)..(1)Xaa may be Ser, Ile, Val or Leumisc_feature(2)..(2)Xaa can be any naturally occurring amino acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino acidMISC_FEATURE(6)..(6)Xaa may be Ile, Val or Leu 63Xaa Xaa Tyr Xaa Xaa Xaa 1 5

Claims

1. A cell which comprises a chimeric antigen receptor (CAR) and a signal transduction modifying protein, selected from one of the following: (i) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase domain; (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain; (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain.

2. (canceled)

3. The cell according to claim 1, wherein the signal transduction modifying protein is a truncated protein which comprises an PTPN6 SH2 but lacks a PTPN6 phosphatase domain.

4. The cell according to claim 1, wherein the signal transduction modifying protein is a truncated protein which comprises a SHP-2 SH2 domain but lacks a SHP-2 phosphatase domain.

5-38. (canceled)

39. A nucleic acid construct, which comprises: a first nucleic acid sequence encoding a chimeric antigen receptor; and a second nucleic acid sequence encoding: (i) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase domain; (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain; or (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain.

40-42. (canceled)

43. A vector which comprises a nucleic acid construct according to claim 39.

44. (canceled)

45. The vector according to claim 43, which is a retroviral or lentiviral vector.

46. A pharmaceutical composition comprising a plurality of cells according to claim 1.

47. (canceled)

48. A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 46 to a subject.

49. The method according to claim 48, which comprises the following steps: (i) isolation of a cell containing sample from a subject; (ii) transduction or transfection of the cells with a nucleic acid construct according to claim 39 or a vector comprising the nucleic acid construct; and (iii) administering the cells from (ii) to the subject.

50. (canceled)

51. The method according to claim 48, wherein the disease is cancer.

52. A method for making a cell according to claim 1, which comprises the step of introducing into the cell: a nucleic acid construct or a vector comprising the nucleic acid construct, wherein the nucleic acid construct comprises a first nucleic acid sequence encoding a chimeric antigen receptor and a second nucleic acid sequence encoding: (i) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase domain; (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain; or (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain.

53. The method according to claim 52, wherein the cell is from a sample isolated from a subject.